Fibroblast growth factor-13

ABSTRACT

The present invention relates to a novel FGF-13 protein which is a member of the fibroblast growth factor (FGF) family. In particular, isolated nucleic acid molecules are provided encoding the human FGF-13 protein. FGF-13 polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of FGF-13 activity. Also provided are diagnostic methods for detecting FGF-13-related disorders and therapeutic methods for treating FGF-13-related disorders. Disclosed is a method of prolonging dopaminergic neuron survival.

This application is a continuation-in-part of, and claims the benefit ofpriority under 35 U.S.C. §120 to U.S. application Ser. No. 08/462,965,filed Jun. 5, 1995, now issued as U.S. Pat. No. 5,728,546 and PCTApplication No. PCT/US095/07108, filed Jun. 5, 1995. This applicationalso claims the benefit of priority under 35 U.S.C. §119(e) of U.S.Provisional Application Ser. Nos. 60/031,969 and 60/031,575, filed Nov.27, 1996 and Dec. 4, 1996, respectively.

FIELD OF THE INVENTION

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptide of the presentinvention have been putatively identified as fibroblast growthfactor/heparin binding growth factor, hereinafter referred to asFibroblast Growth Factor-13 (hereafter, “FGF-13”). The invention alsorelates to inhibiting the action of such polypeptides.

BACKGROUND OF THE INVENTION

Fibroblast growth factors are a family of proteins characteristic ofbinding to heparin and are, therefore, also called heparin bindinggrowth factors (HBGF). Expression of different members of these proteinsare found in various tissues and are under particular temporal andspatial control. These proteins are potent mitogens for a variety ofcells of mesodermal, ectodermal, and endodermal origin, includingfibroblasts, corneal and vascular endothelial cells, granulocytes,adrenal cortical cells, chondrocytes, myoblasts, vascular smooth musclecells, lens epithelial cells, melanocytes, keratinocytes,oligodendrocytes, astrocytes, osteoblasts, and hematopoietic cells.

Each member has functions overlapping with others and also has itsunique spectrum of functions. In addition to the ability to stimulateproliferation of vascular endothelial cells, both FGF-1 and 2 arechemotactic for endothelial cells and FGF-2 has been shown to enableendothelial cells to penetrate the basement membrane. Consistent withthese properties, both FGF-1 and 2 have the capacity to stimulateangiogenesis. Another important feature of these growth factors is theirability to promote wound healing. Many other members of the FGF familyshare similar activities with FGF-1 and 2 such as promoting angiogenesisand wound healing. Several members of the FGF family have been shown toinduce mesoderm formation and to modulate differentiation of neuronalcells, adipocytes and skeletal muscle cells.

Other than these biological activities in normal tissues, FGF proteinshave been implicated in promoting tumorigenesis in carcinomas andsarcomas by promoting tumor vascularization and as transforming proteinswhen their expression is deregulated.

The FGF family presently consists of eight structurally-relatedpolypeptides: basic FGF, acidic FGF, int 2, hst 1/k-FGF, FGF-5, FGF-6,keratinocyte growth factor, AIGF (FGF-8); and recently a glia-activatingfactor has been shown to be a novel heparin-binding growth factor whichwas purified from the culture supernatant of a human glioma cell line(Miyamoto, M. et al., Mol. and Cell. Biol., 13(7):4251-4259 (1993). Thegenes for each have been cloned and sequenced. Two of the members, FGF-1and FGF-2, have been characterized under many names, but most often asacidic and basic fibroblast growth factor, respectively. The normal geneproducts influence the general proliferation capacity of the majority ofmesoderm and neuroectoderm-derived cells. They are capable of inducingangiogenesis in vivo and may play important roles in early development(Burgess, W. H. and Maciag, T., Ann. Rev. Biochem., 58:575-606 (1989)).

Many of the above-identified members of the FGF family also bind to thesame receptors and elicit a second message through binding to thesereceptors.

A eukaryotic expression vector encoding a secreted form of FGF-1 hasbeen introduced by gene transfer into porcine arteries. This modeldefines gene function in the arterial wall in vivo. FGF-1 expressioninduced intimal thickening in porcine arteries 21 days after genetransfer (Nabel, E. G., et al., Nature, 362:844-6 (1993)). It hasfurther been demonstrated that basic fibroblast growth factor mayregulate glioma growth and progression independent of its role in tumorangiogenesis and that basic fibroblast growth factor release orsecretion may be required for these actions (Morrison, R. S., et al., J.Neurosci. Res. 34:502-509 (1993)).

Fibroblast growth factors, such as basic FGF, have further beenimplicated in the growth of Kaposi's sarcoma cells in vitro (Huang, Y.Q., et al., J. Clin. Invest. 91:1191-1197 (1993)). Also, the cDNAsequence encoding human basic fibroblast growth factor has been cloneddownstream of a transcription promoter recognized by the bacteriophageT7 RNA polymerase. Basic fibroblast growth factors so obtained have beenshown to have biological activity indistinguishable from human placentalfibroblast growth factor in mitogenicity, synthesis of plasminogenactivator and angiogenesis assays (Squires, C. H., et al., J. Biol.Chem. 263:16297-16302 (1988)).

U.S. Pat. No. 5,155,214 discloses substantially pure mammalian basicfibroblast growth factors and their production. The amino acid sequencesof bovine and human basic fibroblast growth factor are disclosed, aswell as the DNA sequence encoding the polypeptide of the bovine species.

Newly discovered FGF-9 has around 30% sequence similarity to othermembers of the FGF family. Two cysteine residues and other consensussequences in family members were also well conserved in the FGF-9sequence. FGF-9 was found to have no typical signal sequence in its Nterminus like those in acidic and basic FGF. However, FGF-9 was found tobe secreted from cells after synthesis despite its lack of a typicalsignal sequence FGF (Miyamoto, M. et al., Mol. and Cell. Biol.13(7):4251-4259 (1993). Further, FGF-9 was found to stimulate the cellgrowth of oligodendrocyte type 2 astrocyte progenitor cells, BALB/c 3T3,and PC-12 cells but not that of human umbilical vein endothelial cells(Naruo, K., et al., J. Biol. Chem. 268:2857-2864 (1993).

Basic FGF and acidic FGF are potent modulators of cell proliferation,cell motility, differentiation, and survival and act on cell types fromectoderm, mesoderm and endoderm. These two FGFs, along with KGF andAIGF, were identified by protein purification. However, the other fourmembers were isolated as oncogenes, expression of which was restrictedto embryogenesis and certain types of cancers. FGF-9 was demonstrated tobe a mitogen against glial cells. Members of the FGF family are reportedto have oncogenic potency. FGF-9 has shown transforming potency whentransformed into BALB/c 3T3 cells (Miyamoto, M., et al., Mol. Cell.Biol. 13(7):4251-4259 (1993).

Androgen induced growth factor (AIGF), also known as FGF-8, was purifiedfrom a conditioned medium of mouse mammary carcinoma cells (SC-3)simulated with testosterone. AIGF is a distinctive FGF-like growthfactor, having a putative signal peptide and sharing 30-40% homologywith known members of the FGF family. Mammalian cells transformed withAIGF shows a remarkable stimulatory effect on the growth of SC-3 cellsin the absence of androgen. Therefore, AIGF mediates androgen-inducedgrowth of SC-3 cells, and perhaps other cells, since it is secreted bythe tumor cells themselves.

SUMMARY OF THE INVENTION

The polypeptide of the present invention has been putatively identifiedas a member of the FGF family as a result of amino acid sequencehomology with other members of the FGF family.

In accordance with one aspect of the present invention, there areprovided novel mature polypeptides as well as biologically active anddiagnostically or therapeutically useful fragments, analogs andderivatives thereof. The polypeptides of the present invention are ofhuman origin.

In accordance with another aspect of the present invention, there areprovided isolated nucleic acid molecules encoding the polypeptides ofthe present invention, including mRNAs, DNAs, cDNAs, genomic DNA, aswell as antisense analogs thereof and biologically active anddiagnostically or therapeutically useful fragments thereof.

Thus, the present invention provides -isolated nucleic acid moleculescomprising a polynucleotide encoding at least a portion of the FGF-13polypeptide having the complete amino acid sequence shown in SEQ ID NO:2or the complete amino acid sequence encoded by the cDNA clone depositedas plasmid DNA in a bacterial host as ATCC Deposit Number 97148 on May12, 1995. The nucleotide sequence determined by sequencing the depositedFGF-13 clone, which is shown in FIGS. 1A-D (SEQ ID NO:1), contains anopen reading frame encoding a complete polypeptide of 216 amino acidresidues, including an initiation codon encoding an N-terminalmethionine at nucleotide positions 1 to 3. Nucleic acid molecules of theinvention include those encoding the complete amino acid sequenceexcepting the N-terminal methionine shown in SEQ ID NO:2, or thecomplete amino acid sequence excepting the N-terminal methionine encodedby the cDNA clone in ATCC Deposit Number 97148, which molecules also canencode additional amino acids fused to the N-terminus of the FGF-13amino acid sequence.

Accordingly, one aspect of the invention provides an isolated nucleicacid molecule comprising a polynucleotide comprising a nucleotidesequence selected from the group consisting of: (a) a nucleotidesequence encoding the FGF-13 polypeptide having the complete amino acidsequence in SEQ ID NO:2 excepting the N-terminal methionine (i.e.,positions −22 to 193 of SEQ ID NO:2); (b) a nucleotide sequence encodingthe predicted mature FGF-13 polypeptide having the amino acid sequencefrom about position 1 to about position 193 in SEQ ID NO:2; (c) anucleotide sequence encoding the FGF-13 polypeptide having the completeamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 97148; and (d) a nucleotide sequence encoding the mature FGF-13polypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 97148; and (e) a nucleotide sequencecomplementary to any of the nucleotide sequences in (a), (b), (c) or (d)above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical, to any of the nucleotide sequences in (a), (b), (c), (d)or (e), above, or a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide in (a), (b), (c), (d) or(e), above. This polynucleotide which hybridizes does not hybridizeunder stringent hybridization conditions to a polynucleotide having anucleotide sequence consisting of only A residues or of only T residues.An additional nucleic acid embodiment of the invention relates to anisolated nucleic acid molecule comprising a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion of an FGF-13polypeptide having an amino acid sequence in (a), (b), (c) or (d),above.

In accordance with still another aspect of the present invention, thereare provided processes for producing such polypeptides by recombinanttechniques through the use of recombinant vectors, such as cloning andexpression plasmids useful as reagents in the recombinant production ofthe polypeptides of the present invention, as well as recombinantprokaryotic and/or eukaryotic host cells comprising a nucleic acidsequence encoding a polypeptide of the present invention.

In accordance with a further aspect of the present invention, there isprovided a process for utilizing such polypeptides, or polynucleotidesencoding such polypeptides, for screening for agonists and antagoniststhereto and for therapeutic purposes, for example, promoting woundhealing for example as a result of burns and ulcers, to prevent neuronaldamage associated with stroke and due to neuronal disorders and promoteneuronal growth for example Parkinson's disease, and to prevent skinaging and hair loss, to stimulate angiogenesis, mesodermal induction inearly embryos and limb regeneration.

In accordance with yet a further aspect of the present invention, thereare provided antibodies against such polypeptides.

In accordance with yet another aspect of the present invention, thereare provided antagonists against such polypeptides and processes fortheir use to inhibit the action of such polypeptides, for example, inthe treatment of cellular transformation, for example, tumors, to reducescarring and treat hyper-vascular diseases.

In accordance with another aspect of the present invention, there areprovided nucleic acid probes comprising nucleic acid molecules ofsufficient length to specifically hybridize to a polynucleotide encodinga polypeptide of the present invention.

In another embodiment, the invention provides an isolated antibody thatbinds specifically to an FGF-13 polypeptide having an amino acidsequence described in (a), (b), (c) or (d) above. The invention furtherprovides methods for isolating antibodies that bind specifically to anFGF-13 polypeptide having an amino acid sequence as described herein.Such antibodies are useful diagnostically or therapeutically asdescribed below.

In accordance with yet another aspect of the present invention, thereare provided diagnostic assays for detecting diseases or susceptibilityto diseases related to mutations in a nucleic acid sequence of thepresent invention and for detecting over-expression or under-expressionof the polypeptides encoded by such sequences.

In accordance with another aspect of the present invention, there isprovided a process for utilizing such polypeptides, or polynucleotidesencoding such polypeptides, for in vitro purposes related to scientificresearch, synthesis of DNA and manufacture of DNA vectors. Thus, theinvention also provides pharmaceutical compositions comprising FGF-13polypeptides, particularly human FGF-13 polypeptides. Methods oftreating individuals in need of FGF-13 polypeptides are also provided.The invention further provides compositions comprising an FGF-13polynucleotide or an FGF-13 polypeptide for administration to cells invitro, to cells ex vivo and to cells in vivo, or to a multicellularorganism. In certain particularly preferred embodiments of this aspectof the invention, the compositions comprise an FGF-13 polynucleotide forexpression of an FGF-13 polypeptide in a host organism for treatment ofdisease. Particularly preferred in this regard is expression in a humanpatient for treatment of a dysfunction associated with aberrantendogenous activity of an FGF-13 gene.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings are meant only as illustrations of specificembodiments of the present invention and are not meant as limitations inany manner.

FIGS. 1A-D depicts the nucleotide sequence (SEQ ID NO:1) of the humanmRNA encoding FGF-13 and the deduced amino acid sequence (SEQ ID NO:2)of the FGF-13 polypeptide. The putative leader sequence of about 23amino acids is underlined. Note that the methionine residue at thebeginning of the leader sequence in FIGS. 1A-D is shown in positionnumber (positive) 1, whereas the leader positions in the correspondingsequence of SEQ ID NO:2 are designated with negative position numbers.Thus, the leader sequence positions 1 to 23 in FIGS. 1A-D correspond topositions −23 to −1 in SEQ ID NO:2.

FIG. 2 shows an alignment of the regions of identity among the aminoacid sequences of the human FGF-13 protein and the amino acid sequencesof the following human proteins: acidic FGF (SEQ ID NO:3), basic FGF(SEQ ID NO:4), Int-2 (SEQ ID NO:5), FGF-4 (SEQ ID NO:6), FGF-5 (SEQ IDNO:7), FGF-6 (SEQ ID NO:8), Keratinocyte Growth Factor (KGF) (SEQ IDNO:9), and AIGF (FGF-8) (SEQ ID NO:10), as determined by the “Megalign”routine of the DNAStar program.

FIG. 3 shows an analysis of the FGF-13 amino acid sequence. Alpha, beta,turn and coil regions; hydrophilicity and hydrophobicity; amphipathicregions; flexible regions; antigenic index and surface probability areshown. In the “Antigenic Index—Jameson-Wolf” graph, the indicatelocation of the highly antigenic regions of the FGF-13 protein, i.e.,regions from which epitope-bearing peptides of the invention may beobtained.

FIG. 4. FGF-13 treatment increases the number of cells in embryoniccortical cultures. The cells, derived from gestation day 17 embryos,were plated in polylysine/laminin coated wells at a density of 354cells/mm². The cultures were maintained in serum-free medium and treatedevery other day with the indicated concentrations of FGFs. Aftertreatment for 7-8 days, the cell number was estimated by labeling thecultures with Calcein AM and monitoring the level of fluorescenceemission at 530 nm. The data points represent the mean of 5-6determinations±the standard error. (Key: =FGF-13; =bFGF(rh)).

FIG. 5. The increase in high-affinity neuronal specific GABA-uptakeinduced by FGF-13 is enhanced by heparin. The cortical cultures, platedat a density of 1770 cells/mm2 in poly-lysine/laminin coated wells, weretreated for 7-8 days with FGF-13 in the presence or absence of heparin.The heparin and FGF-13 were pre-incubated for approximately 30 min priorto addition to the cultures. The data points represent the means of 4determinations±the standard error. (Key: ▪=FGF-13; =FGF-13+10 ng/mlheparin; ▴=FGF-13+100 ng/ml heparin).

FIG. 6. FGF-13 treatment enhances neurite outgrowth of cortical neurons.For the neurite outgrowth experiments, the cultures were plated at adensity of 212 cell/mm2 on poly-lysine coated wells. The cultures werethen fixed and the amount of the 68 kDa neurofilament subunit wasdetermined by ELISA. The data points are the means of 5-6determinations±the standard error . . . (Key: =FGF-13; =bFGF(rh)).

FIG. 7. FGF-13 induces the proliferation of rat hippocampal astrocytes.The astrocytes were sub-cultured at a density of 15,000 cells/well in 96well plates. The cells were arrested in G1 phase by an 18 hr incubationin serum-free medium and then treated with FGF-13 in the absence orpresence of heparin for 24 hours. During the last 4 hr of the incubationperiod, the cultures were labeled with [³H]-thymidine. The data pointsrepresent the mean of 4-6 determinations±the standard error . . . (Key:=FGF-13; =FGF-13+10 ng/ml heparin; =FGF-13+100 ng/ml heparin;▪=FGF-13+1000 ng/ml heparin).

FIG. 8. FGF-13 displaces the binding of [¹²⁵I]FGF-1 from monolayercultures of hippocampal astrocytes. Astrocytes were subcultured andgrown to confluence in 24 well plates. The cultures were incubated at 4°C. with 50 pM [¹²⁵I]FGF-1 in the absence or presence of the indicatedconcentration of unlabeled FGFs. The data points are the means of 4determinations±the standard error. (Key: =FGF-10; =bFGF; ▪=FGF-13).

FIG. 9. Effect of FGF-13 treatment on human lung fibroblastproliferation. (Key: ▪=bFGF; ♦=FGF-13).

FIG. 10. Effect of FGF-13 treatment on human dermal endothelial cellproliferation . . . (Key: ▪=bFGF; ♦FGF-13).

FIG. 11. Effect of FGF-2 and FGF-13 on release of PGE₂ from human lungfibroblasts. (Key: in each group of six bars, from left to right thebars represent: medium alone; bFGF (FGF-2) (100 ng.ml); indomethicin(100 ng/ml); FGF-13 (100 ng/ml); FGF-13 (1000 ng/ml); FGF-12 (2500ng/ml)).

FIG. 12. Effects of FGF-2 and FGF-13 on release of IL-6 from human lungfibroblasts. (Key: in each group of six bars, from left to right thebars represent: medium alone; bFGF (FGF-2) (100 ng.ml); indomethicin(100 ng/ml); FGF-13 (100 ng/ml); FGF-13 (1000 ng/ml); FGF-12 (2500ng/ml)).

FIG. 13 shows mitogenic activity of FGF13 on BaF3 cells expressing FGFreceptors (FGFR) 1c, 2c, 3c, and 4 measured by thymidine incorporation.BaF3 cells expressing FGF receptors were incubated with FGF-13 up toconcentration of 62.5 nM (X axis). aFGF at the same concentration rangewas used as a positive control. The Y axis represents the amount of ³Hthymidine incorporated into DNA of BaF3 cells as a percentage of themaximum cpm incorporated following FGF1 stimulation. Circle: FGFR1c;Square: FGFR2c; Diamond: FGFR3c; Triangle: FGFR4.

FIG. 14 shows the tyrosine hydroxylase stimulatory activity of FGF13 oncultured dopaminergic neurons from the midbrain floor dissected from E14Wistar rat embryos, dissociated with trypsin and seeded at a density of200,000 cells per square centimeter. Tyrosine hydroxylase positiveneurons increased after FGF-13 administration. The control was basicFGF, other fibroblase growth factors include FGF-2, FGF-9, and FGF-10.

DETAILED DESCRIPTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding an FGF-13 polypeptide having theamino acid sequence shown in SEQ ID NO:2, which was determined bysequencing cloned cDNAs. The nucleotide sequence shown in positions11-1212 of SEQ ID NO:1 was obtained by sequencing the HODAH63 clone,which was deposited on May 12, 1995 at the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209, andgiven accession number ATCC 97148. The deposited clone is contained inthe pBluescript SK(−) plasmid (Stratagene, La Jolla, Calif.). Thenucleotide sequence shown in positions 1-10 of SEQ ID NO:1 was obtainedby sequencing the product of a PCR amplification of a cDNA librarycontaining a mixture of human cDNAs.

The deposit referred to herein will be maintained under the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the purposes of Patent Procedure. This deposit is provided merely asa convenience and is not an admission that a deposit is required under35 U.S.C. §112. The sequence of the polynucleotides contained in thedeposited materials, as well as the amino acid sequence of thepolypeptides encoded thereby, are incorporated herein by reference andare controlling in the event of any conflict with the description ofsequences herein. A license may be required to make, use or sell thedeposited materials, and no such license is hereby granted.

The FGF-13 polypeptide is structurally related to all members of thefibroblast growth factor family and contains an open reading frameencoding a polypeptide of 216 amino acids (SEQ ID NO:2) of which thefirst 23 amino acids represent a putative leader sequence such that themature polypeptide comprises 193 amino acids. Among the top matches are:

1) 69% identity and 81% similarity to mouse AIGF over a stretch of 185amino acids; 2) 30% identity and 56% similarity with FGF-4 from in aregion of 82 amino acids; 3) 41% identity and 64% similarity with humanKGF (SEQ ID NO:9) over a stretch of 78 amino acids. Among human homologscompared to FGF-13, FGF-8 (bFGF) and aFGF show the greatest similarities(56.7% and 51.0%, respectively). An alignment of the FGF-13 amino acidsequence with the amino acids sequences of various human polypeptides isshown in FIG. 2.

The FGF/HBGF family signature, GXLX(S, T, A, G)X6(D, E)CXFXE (SEQ IDNO:38), is conserved in the polypeptide of the present invention (Xmeans any amino acid residue; (D, E) means either D or E residue; X6means any 6 amino acid residues).

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc., FosterCity, Calif.), and all amino acid sequences of polypeptides encoded byDNA molecules determined herein were predicted by translation of a DNAsequence determined as above. Therefore, as is known in the art for anyDNA sequence determined by this automated approach, any nucleotidesequence determined herein may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90% identical,more typically at least about 95% to at least about 99.9% identical tothe actual nucleotide sequence of the sequenced DNA molecule. The actualsequence can be more precisely determined by other approaches includingmanual DNA sequencing methods well known in the art. As is also known inthe art, a single insertion or deletion in a determined nucleotidesequence compared to the actual sequence will cause a frame shift intranslation of the nucleotide sequence such that the predicted aminoacid sequence encoded by a determined nucleotide sequence will becompletely different from the amino acid sequence actually encoded bythe sequenced DNA molecule, beginning at the point of such an insertionor deletion.

By “nucleotide sequence” of a nucleic acid molecule or polynucleotide isintended, for a DNA molecule or polynucleotide, a sequence ofdeoxyribonucleotides, and for an RNA molecule or polynucleotide, thecorresponding sequence of ribonucleotides (A, G, C and U), where eachthymidine deoxyribonucleotide (T) in the specified deoxyribonucleotidesequence is replaced by the ribonucleotide uridine (U).

Using the information provided herein, such as the nucleotide sequencein FIGS. 1A-D (SEQ ID NO:1), a nucleic acid molecule of the presentinvention encoding an FGF-13 polypeptide may be obtained using standardcloning and screening procedures, such as those for cloning cDNAs usingmRNA as starting material. Illustrative of the invention, the nucleicacid molecule described in FIGS. 1A-D (SEQ ID NO:1) was discovered in acDNA library derived from human ovarian cancer tissue. Additional clonesof the same gene were also identified in cDNA libraries from human fetalkidney tissue, and Northern blotting of human tissues detected a weaksignal (1.6 kb) only in human fetal kidney and human fetal brain.Therefore, nucleic acids of the invention are useful as hybridizationprobes for differential identification of the tissue(s) or cell type(s)present in a biological sample. Similarly, polypeptides and antibodiesdirected to those polypeptides are useful to provide immunologicalprobes for differential identification of tissue(s) or cell type(s).

The determined nucleotide sequence of the FGF-13 cDNA of FIGS. 1A-D (SEQID NO:1) contains an open reading frame encoding a protein of 216 aminoacid residues, with an initiation codon at nucleotide positions 1 to 3of the nucleotide sequence in FIGS. 1A-D (SEQ ID NO:1). The FGF-13polypeptide encoded by the deposited cDNA actually comprises about the212 amino acids at the C-terminal end of the sequence in SEQ ID NO:2,with the remaining N-terminal sequences in FIGS. 1A-D and SEQ ID NO:2having been determined by sequencing a product of a polymerase chainreaction (PCR) mixture using DNA from a human cDNA library in a phagevector for the template, primed by vector-specific primers 5′ and 3′ tothe cDNA insert site. As one of ordinary skill would appreciate, due tothe possibilities of sequencing errors discussed above, the actualFGF-13 polypeptide encoded by the deposited cDNA, which comprises about212 amino acids at the C-terminal end of the sequence in SEQ ID NO:2,may be somewhat longer or shorter than the determined sequence. Moregenerally, the actual open reading frame may be anywhere in the range of±20 amino acids, more likely in the range of ±10 amino acids, of thatpredicted from the N-terminus shown in FIGS. 1A-D (SEQ ID NO:1).

Leader and Mature Sequences

The amino acid sequence of the complete FGF-13 protein includes a leadersequence and a mature protein, as shown in SEQ ID NO:2. More inparticular, the present invention provides nucleic acid moleculesencoding a mature form of the FGF-13 protein. Thus, according to thesignal hypothesis, once export of the growing protein chain across therough endoplasmic reticulum has been initiated, proteins secreted bymammalian cells have a signal or secretory leader sequence which iscleaved from the complete polypeptide to produce a secreted “mature”form of the protein. Most mammalian cells and even insect cells cleavesecreted proteins with the same specificity. However, in some cases,cleavage of a secreted protein is not entirely uniform, which results intwo or more mature species of the protein. Further, it has long beenknown that the cleavage specificity of a secreted protein is ultimatelydetermined by the primary structure of the complete protein, that is, itis inherent in the amino acid sequence of the polypeptide.

Therefore, the present invention provides a nucleotide sequence encodingthe mature FGF-13 polypeptide having the amino acid sequence encoded bythe cDNA clone contained in the host identified as ATCC Deposit No.97148. By the “mature FGF-13 polypeptide having the amino acid sequenceencoded by the cDNA clone in ATCC Deposit No. 97148” is meant the matureform(s) of the FGF-13 protein produced by expression in a mammalian cell(e.g., COS cells, as described below) by a DNA encoding the completeFGF-13 coding sequence encoded by the human DNA sequence of the clonecontained in the vector in the deposited host, when that human DNAsequence is operably linked to appropriate regulatory sequences fortranslation of the FGF-13 coding sequence including an initiation codon.

In the present case, the deduced amino acid sequence of the completeFGF-13 polypeptide was analyzed by alignment with the known amino acidsequence of human FGF-8 (see FIG. 2), thereby predicting a secretoryleader cleavage site at the homologous location within the completeamino acid sequence shown in SEQ ID NO:2, between residues −1 and +1.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising an open reading frame (ORF) with or without aninitiation codon at positions 1 to 3 of the nucleotide sequence shown inFIGS. 1A-D (SEQ ID NO:1). Also included are DNA molecules comprising thecoding sequence for the predicted mature FGF-13 protein shown atpositions 1 to 193 of SEQ ID NO:2.

In addition, isolated nucleic acid molecules of the invention includeDNA molecules which comprise a sequence substantially different fromthose described above but which, due to the degeneracy of the geneticcode, still encode an FGF-13 protein. Of course, the genetic code andspecies-specific codon preferences are well known in the art. Thus, itwould be routine for one skilled in the art to generate the degeneratevariants described above, for instance, to optimize codon expression fora particular host (e.g., change codons in the human mRNA to thosepreferred by a bacterial host such as E. coli).

In another aspect, the invention provides isolated nucleic acidmolecules encoding the FGF-13 polypeptide having an amino acid sequenceencoded by the cDNA clone contained in the plasmid deposited as ATCCDeposit No. 97148 on May 12, 1995. Preferably, this nucleic acidmolecule will encode the mature polypeptide encoded by theabove-described deposited cDNA clone.

The invention further provides an isolated nucleic acid molecule havingthe nucleotide sequence shown in FIGS. 1A-D (SEQ ID NO:1) or thenucleotide sequence of the FGF-13 cDNA contained in the above-describeddeposited clone, or a nucleic acid molecule having a sequencecomplementary to one of the above sequences. Such isolated molecules,particularly DNA molecules, are useful as probes for gene mapping, by insitu hybridization with chromosomes, and for detecting expression of theFGF-13 gene in human tissue, for instance, by Northern blot analysis.

The present invention is further directed to nucleic acid moleculesencoding portions of the nucleotide sequences described herein as wellas to fragments of the isolated nucleic acid molecules described herein.In particular, the invention provides a polynucleotide having anucleotide sequence representing the portion of SEQ ID NO:1 whichconsists of positions 1 to 648 of SEQ ID NO:1.

Thus, the invention includes a polynucleotide comprising any portion ofat least about 30 nucleotides, preferably at least about 50 nucleotides,of SEQ ID NO:1 from residue 1 to 648. More generally, by a fragment ofan isolated nucleic acid molecule having the nucleotide sequence of thedeposited cDNA or the nucleotide sequence shown in FIGS. 1A-D (SEQ IDNO:1) is intended fragments at least about 15 nt, and more preferably atleast about 20 nt, still more preferably at least about 30 nt, and evenmore preferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments 50-300 nt in length are also useful according to the presentinvention as are fragments corresponding to most, if not all, of thenucleotide sequence of the deposited cDNA or as shown in FIGS. 1A-D (SEQID NO:1). By a fragment at least 20 nt in length, for example, isintended fragments which include 20 or more contiguous bases from thenucleotide sequence of the deposited cDNA or the nucleotide sequence asshown in FIGS. 1A-D (SEQ ID NO:1). Preferred nucleic acid fragments ofthe present invention include nucleic acid molecules encodingepitope-bearing portions of the FGF-13 polypeptide as identified in FIG.3 and described in more detail below.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, the cDNAclone contained in ATCC Deposit No. 97148. By “stringent hybridizationconditions” is intended overnight incubation at 42° C. in a solutioncomprising: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed bywashing the filters in 0.1×SSC at about 65° C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 (e.g., 50) nt of the reference polynucleotide. These areuseful as diagnostic probes and primers as discussed above and in moredetail below.

By a portion of a polynucleotide of “at least 20 nt in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in FIGS. 1A-D (SEQ ID NO:1)).Of course, a polynucleotide which hybridizes only to a poly A sequence(such as the 3′ terminal poly(A) tract of the FGF-13 cDNA shown in FIGS.1A-D (SEQ ID NO:1)), or to a complementary stretch of T (or U) residues,would not be included in a polynucleotide of the invention used tohybridize to a portion of a nucleic acid of the invention, since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

As indicated, nucleic acid molecules of the present invention whichencode an FGF-13 polypeptide may include, but are not limited to thoseencoding the amino acid sequence of the mature polypeptide, by itself;and the coding sequence for the mature polypeptide and additionalsequences, such as those encoding the about 25 amino acid leader orsecretory sequence, such as a pre-, or pro- or prepro-protein sequence;the coding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences.

Also encoded by nucleic acids of the invention are the above proteinsequences together with additional, non-coding sequences, including forexample, but not limited to introns and non-coding 5′ and 3′ sequences,such as the transcribed, non-translated sequences that play a role intranscription, mRNA processing, including splicing and polyadenylationsignals, for example—ribosome binding and stability of mRNA; anadditional coding sequence which codes for additional amino acids, suchas those which provide additional functionalities.

Thus, the sequence encoding the polypeptide may be fused to a markersequence, such as a sequence encoding a peptide which facilitatespurification of the fused polypeptide. In certain preferred embodimentsof this aspect of the invention, the marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson et al., Cell 37: 767 (1984).As discussed below, other such fusion proteins include the FGF-13 fusedto Fc at the N- or C-terminus.

Variant and Mutant Polynucleotides

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the FGF-13 protein. Variants may occur naturally, such asa natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. The variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the FGF-13 protein or portions thereof.Also especially preferred in this regard are conservative substitutions.

Most highly preferred are nucleic acid molecules encoding the matureprotein having the amino acid sequence shown in SEQ ID NO:2 or themature FGF-13 amino acid sequence encoded by the deposited cDNA clone.

Further embodiments include an isolated nucleic acid molecule comprisinga polynucleotide having a nucleotide sequence at least 90% identical,and more preferably at least 95%, 96%, 97%, 98% or 99% identical to apolynucleotide selected from the group consisting of: (a) a nucleotidesequence encoding the FGF-13 polypeptide having the complete amino acidsequence in SEQ ID NO:2; (b) a nucleotide sequence encoding thepredicted mature FGF-13 polypeptide having the amino acid sequence atpositions 1 to 193 of SEQUENCE ID NO:2; (c) a nucleotide sequenceencoding the FGF-13 polypeptide having the complete amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 97148; (d) anucleotide sequence encoding the mature FGF-13 polypeptide having theamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo. 97148; and (e) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c) or (d) above.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding an FGF-13polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the FGF-13polypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5′ or 3′ terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequence shown in FIG. 1 or to the nucleotides sequence ofthe deposited cDNA clone can be determined conventionally using knowncomputer programs such as the Bestfit program (Wisconsin SequenceAnalysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711).Bestfit uses the local homology algorithm of Smith and Waterman,Advances in Applied Mathematics 2:482-489 (1981), to find the bestsegment of homology between two sequences. When using Bestfit or anyother sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

The present application is directed to nucleic acid molecules at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIG. 1 (SEQ ID NO:1) or to the nucleic acid sequence of thedeposited cDNA, irrespective of whether they encode a polypeptide havingFGF-13 activity. This is because even where a particular nucleic acidmolecule does not encode a polypeptide having FGF-13 activity, one ofskill in the art would still know how to use the nucleic acid molecule,for instance, as a hybridization probe or a polymerase chain reaction(PCR) primer. Uses of the nucleic acid molecules of the presentinvention that do not encode a polypeptide having FGF-13 activityinclude, inter alia, (1) isolating the FGF-13 gene or allelic variantsthereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) tometaphase chromosomal spreads to provide precise chromosomal location ofthe FGF-13 gene, as described in Verna et al., Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York (1988); andNorthern blot analysis for detecting FGF-13 mRNA expression in specifictissues.

Preferred, however, are nucleic acid molecules having sequences at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIG. 1 (SEQ ID NO:1) or to the nucleic acid sequence of thedeposited cDNA which do, in fact, encode a polypeptide having FGF-13protein activity. By “a polypeptide having FGF-13 activity” is intendedpolypeptides exhibiting activity similar, but not necessarily identical,to an activity of the mature FGF-13 protein of the invention, asmeasured in a particular biological assay. For example, the FGF-13protein of the present invention stimulates proliferations of variousmammalian cells, particularly fibroblasts, as described further below.

FGF-13 protein stimulates cellular proliferation in a dose-dependentmanner in the various activity assays described hereinbelow. Thus, “apolypeptide having FGF-13 protein activity” includes polypeptides thatalso exhibit any of the same activities in the below-described assays ina dose-dependent manner. Although the degree of dose-dependent activityneed not be identical to that of the FGF-13 protein, preferably, “apolypeptide having FGF-13 protein activity” will exhibit substantiallysimilar dose-dependence in a given activity as compared to the FGF-13protein (i.e., the candidate polypeptide will exhibit greater activityor not more than about 25-fold less and, preferably, not more than abouttenfold less activity relative to the reference FGF-13 protein).

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the deposited cDNAor the nucleic acid sequence shown in FIG. 1 (SEQ ID NO:1) will encode apolypeptide “having FGF-13 protein activity.” In fact, since degeneratevariants of these nucleotide sequences all encode the same polypeptide,this will be clear to the skilled artisan even without performing theabove described comparison assay. It will be further recognized in theart that, for such nucleic acid molecules that are not degeneratevariants, a reasonable number will also encode a polypeptide havingFGF-13 protein activity. This is because the skilled artisan is fullyaware of amino acid substitutions that are either less likely or notlikely to significantly effect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid), as furtherdescribed below.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, and the production of FGF-13polypeptides or fragments thereof by recombinant techniques. The vectormay be, for example, a phage, plasmid, viral or retroviral vector.Retroviral vectors may be replication competent or replicationdefective. In the latter case, viral propagation generally will occuronly in complementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp, phoA and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe transcripts expressed by the constructs will preferably include atranslation initiating codon at the beginning and a termination codon(UAA, UGA or UAG) appropriately positioned at the end of the polypeptideto be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, 293 and Bowes melanoma cells; andplant cells. Appropriate culture mediums and conditions for theabove-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from QIAGEN, Inc., supra; pBS vectors, Phagescriptvectors, Bluescript vectors, pNH8A, pNH6a, pNH18A, pNH46A, availablefrom Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO,pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV,pMSG and pSVL available from Pharmacia. Other suitable vectors will bereadily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to stabilize andpurify proteins. For example, EP-A-O 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobulin molecules together with another humanprotein or part thereof. In many cases, the Fc part in a fusion proteinis thoroughly advantageous for use in therapy and diagnosis and thusresults, for example, in improved pharmacokinetic properties (EP-A 0232262). On the other hand, for some uses it would be desirable to be ableto delete the Fc part after the fusion protein has been expressed,detected and purified in the advantageous manner described. This is thecase when Fc portion proves to be a hindrance to use in therapy anddiagnosis, for example when the fusion protein is to be used as antigenfor immunizations. In drug discovery, for example, human proteins, suchas hIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995) and K.Johanson et al, J. Biol. Chem. 270:9459-9471 (1995).

The FGF-13 protein can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Polypeptides of the presentinvention include: products purified from natural sources, includingbodily fluids, tissues and cells, whether directly isolated or cultured;products of chemical synthetic procedures; and products produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, bacterial, yeast, higher plant, insect and mammalian cells.Depending upon the host employed in a recombinant production procedure,the polypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes. Thus, it is well known in the artthat the N-terminal methionine encoded by the translation initiationcodon generally is removed with high efficiency from any protein aftertranslation in all eukaryotic cells. While the N-terminal methionine onmost proteins also is efficiently removed in most prokaryotes, for someproteins this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked.

Polypeptides and Fragments

The invention further provides an isolated FGF-13 polypeptide comprisingthe amino acid sequence encoded by the deposited cDNA, or the amino acidsequence in SEQ ID NO:2, or a peptide or polypeptide comprising aportion of the above polypeptides.

Variant and Mutant Polypeptides

To improve or alter the characteristics of FGF-13 polypeptides, proteinengineering may be employed. Recombinant DNA technology known to thoseskilled in the art can be used to create novel mutant proteins or“muteins including single or multiple amino acid substitutions,deletions, additions or fusion proteins. Such modified polypeptides canshow, e.g., enhanced activity or increased stability. In addition, theymay be purified in higher yields and show better solubility than thecorresponding natural polypeptide, at least under certain purificationand storage conditions.

N-Terminal and C-Terminal Deletion Mutants

For instance, for many proteins, including the extracellular domain of amembrane associated protein or the mature form(s) of a secreted protein,it is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. For instance, Ron et al., J. Biol. Chem., 268:2984-2988 (1993)reported modified KGF proteins that had heparin binding activity even if3, 8, or 27 amino-terminal amino acid residues were missing. In thepresent case, polypeptides having deletions of up to about 10 additionalN-terminal residues beyond the predicted leader cleavage point (i.e., upto the Asparagine at position 10 in SEQ ID NO:2) can retain somebiological activity such as cell proliferation stimulating or receptorbinding activity.

However, even if deletion of one or more amino acids from the N-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete or mature from of theprotein generally will be retained when less than the majority of theresidues of the complete or mature protein are removed from theN-terminus. Whether a particular polypeptide lacking N-terminal residuesof a complete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the amino terminus of the amino acidsequence of the FGF-13 shown in SEQ ID NO:2, up to the Asparagine atposition 10. In particular, the present invention provides polypeptidescomprising the amino acid sequence of residues n-193 of SEQ ID NO:2,where n is an integer other than zero in the range of −23 to +10(excepting zero). More in particular, the invention providespolynucleotides encoding polypeptides having the amino acid sequence ofresidues of −23 to 193, −22 to 193, −21 to 193, −20 to 193, −19 to 193,−18 to 193, −17 to 193, −16 to 193, −15 to 193, −14 to 193, −13 to 193,−12 to 193, −11 to 193, −10 to 193, 31 9 to 193, −8 to 193, −7 to 193,−6 to 193, −5 to 193, −4 to 193, −3 to 193, −2 to 193, −1 to 193, 1 to193, 2 to 193, 3 to 193, 4 to 193, 5 to 193, 6 to 193, 7 to 193, 8 to193, 9 to 193, and 10 to 193 of SEQ ID NO:2. Polynucleotides encodingthese polypeptides also are provided.

Similarly, many examples of biologically functional C-terminal deletionmuteins are known. For instance, interferon gamna shows up to ten timeshigher activities by deleting 8-10 amino acid residues from the carboxyterminus of the protein (Döbeli et al., J. Biotechnology 7:199-216(1988). In the present case, since the protein of the invention ishomolgous to human FGF-8, deletions of C-terminal amino acids up to theconserved region beginning with the Leucine at position 154 in SEQ IDNO:2 can retain some biological activity such as stimulation of cellularproliferation or receptor binding.

However, even if deletion of one or more amino acids from the C-terminusof a protein results in modification of loss of one or more biologicalfunctions of the protein, other biological activities may still beretained. Thus, the ability of the shortened protein to induce and/orbind to antibodies which recognize the complete or mature form of theprotein generally will be retained when less than the majority of theresidues of the complete or mature protein are removed from theC-terminus. Whether a particular polypeptide lacking C-terminal residuesof a complete protein retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art.

Accordingly, the present invention further provides polypeptides havingone or more residues from the carboxy terminus of the amino acidsequence of the FGF-3 shown in SEQ ID NO:2, up to the Leucine atposition 154 of SEQ ID NO:2, and polynucleotides encoding suchpolypeptides. In particular, the present invention provides polypeptideshaving the amino acid sequence of residues −22 to m of the amino acidsequence in SEQ ID NO:2, where m is any integer in the range of 154 to192, and residue 154 is the position of the first residue from theC-terminus of the complete FGF-13 polypeptide (shown in SEQ ID NO:2)which begins the region highly conserved between FGF-13 and human FGF-8.

More in particular, the invention provides polynucleotides encodingpolypeptides having the amino acid sequence of residues −22 to 154, −22to 155, −22 to 156, −22 to 157, −22 to 158, −22 to 159, −22 to 160, −22to 161, −22 to 162, −22 to 163, −22 to 164, −22 to 165, −22 to 166, −22to 167, −22 to 168, −22 to 169, −22 to 170, −22 to 171, −22 to 172, −22to 173, −22 to 174, −22 to 175, −22 to 176, −22 to 177, −22 to 178, −22to 179, −22 to 180, −22 to 181, −22 to 182, −22 to 183, −22 to 184, −2to 185, −22 to 186, −22 to 187, −22 to 188, −22 to 189, −22 to 190, and−22 to 192 of SEQ ID NO:2. Polynucleotides encoding these polypeptidesalso are provided.

The invention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini, which may bedescribed generally as having residues n-m of SEQ ID NO:2, where n and mare integers as described above.

Also included are a nucleotide sequence encoding a polypeptideconsisting of a portion of the complete FGF-13 amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 97148, wherethis portion excludes from 1 to about 33 amino acids from the aminoterminus of the complete amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 97148, or from 1 to about 39 amino acidsfrom the carboxy terminus, or any combination of the above aminoterminal and carboxy terminal deletions, of the complete amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No. 97148.Polynucleotides encoding all of the above deletion mutant polypeptideforms also are provided.

Other Mutants

In addition to terminal deletion forms of the protein discussed above,it also will be recognized by one of ordinary skill in the art that someamino acid sequences of the FGF-13 polypeptide can be varied withoutsignificant effect of the structure or function of the protein. If suchdifferences in sequence are contemplated, it should be remembered thatthere will be critical areas on the protein which determine activity.

Thus, the invention further includes variations of the FGF-13polypeptide which show substantial FGF-13 polypeptide activity or whichinclude regions of FGF-13 protein such as the protein portions discussedbelow. Such mutants include deletions, insertions, inversions, repeats,and type substitutions selected according to general rules known in theart so as have little effect on activity. For example, guidanceconcerning how to make phenotypically silent amino acid substitutions isprovided in Bowie, J. U. et al., “Deciphering the Message in ProteinSequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310(1990), wherein the authors indicate that there are two main approachesfor studying the tolerance of an amino acid sequence to change. Thefirst method relies on the process of evolution, in which mutations areeither accepted or rejected by natural selection. The second approachuses genetic engineering to introduce amino acid changes at specificpositions of a cloned gene and selections or screens to identifysequences that maintain functionality.

As the authors state, these studies have revealed that proteins aresurprisingly tolerant of amino acid substitutions. The authors furtherindicate which amino acid changes are likely to be permissive at acertain position of the protein. For example, most buried amino acidresidues require nonpolar side chains, whereas few features of surfaceside chains are generally conserved. Other such phenotypically silentsubstitutions are described in Bowie, J. U. et al., supra, and thereferences cited therein. Typically seen as conservative substitutionsare the replacements, one for another, among the aliphatic amino acidsAla, Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

Thus, the fragment, derivative or analog of the polypeptide of SEQ IDNO:2, or that encoded by the deposited cDNA, may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the above form of the polypeptide, such as an IgG Fc fusionregion peptide or leader or secretory sequence or a sequence which isemployed for purification of the above form of the polypeptide or aproprotein sequence. Such fragments, derivatives and analogs are deemedto be within the scope of those skilled in the art from the teachingsherein

Thus, the FGF-13 of the present invention may include one or more aminoacid substitutions, deletions or additions, either from naturalmutations or human manipulation. As indicated, changes are preferably ofa minor nature, such as conservative amino acid substitutions that donot significantly affect the folding or activity of the protein (seeTable 1).

TABLE 1 Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Amino acids in the FGF-13 protein of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro or in vitro proliferativeactivity.

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids which may produce proteins with highlydesirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36:838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).

Replacement of amino acids can also change the selectivity of thebinding of a ligand to cell surface receptors. For example, Ostade etal., Nature 361:266-268 (1993) describes certain mutations resulting inselective binding of TNF-α to only one of the two known types of TNFreceptors. Sites that are critical for ligand-receptor binding can alsobe determined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of the FGF-13 polypeptide can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988). Polypeptides of the invention also can bepurified from natural or recombinant sources using anti-FGF-13antibodies of the invention in methods which are well known in the artof protein purification.

The invention further provides an isolated FGF-13 polypeptide comprisingan amino acid sequence selected from the group consisting of: (a) theamino acid sequence of the complete FGF-13 polypeptide shown in SEQ IDNO:2 excepting the N-terminal methionine (i.e., positions −22 to 193 ofSEQ ID NO:2); (b) the amino acid sequence of the predicted mature FGF-13polypeptide from about position 1 to about position 193 in SEQ ID NO:2;(c) the amino acid sequence of the FGF-13 polypeptide having thecomplete amino acid sequence encoded by the cDNA clone contained in ATCCDeposit No. 97148; and (d) the amino acid sequence of the mature FGF-13polypeptide having the amino acid sequence encoded by the cDNA clonecontained in ATCC Deposit No. 97148.

Further polypeptides of the present invention include polypeptides whichhave at least 90% similarity, more preferably at least 95% similarity,and still more preferably at least 96%, 97%, 98% or 99% similarity tothose described above. The polypeptides of the invention also comprisethose which are at least 80% identical, more preferably at least 90% or95% identical, still more preferably at least 96%, 97%, 98% or 99%identical to the polypeptide encoded by the deposited cDNA or to thepolypeptide of SEQ ID NO:2, and also include portions of suchpolypeptides with at least 30 amino acids and more preferably at least50 amino acids.

By “% similarity” for two polypeptides is intended a similarity scoreproduced by comparing the amino acid sequences of the two polypeptidesusing the Bestfit program (Wisconsin Sequence Analysis Package, Version8 for Unix, Genetics Computer Group, University Research Park, 575Science Drive, Madison, Wis. 53711) and the default settings fordetermining similarity. Bestfit uses the local homology algorithm ofSmith and Waterman (Advances in Applied Mathematics 2:482-489, 1981) tofind the best segment of similarity between two sequences.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of an FGF-13polypeptide is intended that the amino acid sequence of the polypeptideis identical to the reference sequence except that the polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the reference amino acid of the FGF-13 polypeptide. Inother words, to obtain a polypeptide having an amino acid sequence atleast 95% identical to a reference amino acid sequence, up to 5% of theamino acid residues in the reference sequence may be deleted orsubstituted with another amino acid, or a number of amino acids up to 5%of the total amino acid residues in the reference sequence may beinserted into the reference sequence: These alterations of the referencesequence may occur at the amino or carboxy terminal positions of thereference amino acid sequence or anywhere between those terminalpositions, interspersed either individually among residues in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in SEQ ID NO:2 or to the amino acid sequence encodedby deposited cDNA clone can be determined conventionally using knowncomputer programs such the Bestfit program (Wisconsin Sequence AnalysisPackage, Version 8 for Unix, Genetics Computer Group, UniversityResearch Park, 575 Science Drive, Madison, Wis. 53711). When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference amino acid sequence and that gaps in homology ofup to 5% of the total number of amino acid residues in the referencesequence are allowed.

The polypeptide of the present invention could be used as a molecularweight marker on SDS-PAGE gels or on molecular sieve gel filtrationcolumns using methods well known to those of skill in the art.

As described in detail below, the polypeptides of the present inventioncan also be used to raise polyclonal and monoclonal antibodies, whichare useful in assays for detecting FGF-13 protein expression asdescribed below or as agonists and antagonists capable of enhancing orinhibiting FGF-13 protein function. Further, such polypeptides can beused in the yeast two-hybrid system to “capture” FGF-13 protein bindingproteins which are also candidate agonists and antagonists according tothe present invention. The yeast two hybrid system is described inFields and Song, Nature 340:245-246 (1989).

Epitope-Bearing Portions

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide of the invention. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. On the other hand, a region of aprotein molecule to which an antibody can bind is defined as an“antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl Acad. Sci. USA 81:3998-4002 (1983).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Learner, R. A. (1983) “Antibodies that react withpredetermined sites on proteins,” Science, 219:660-666. Peptides capableof eliciting protein-reactive sera are frequently represented in theprimary sequence of a protein, can be characterized by a set of simplechemical rules, and are confined neither to immunodominant regions ofintact proteins (i.e., immunogenic epitopes) nor to the amino orcarboxyl terminals. Antigenic epitope-bearing peptides and polypeptidesof the invention are therefore useful to raise antibodies, includingmonoclonal antibodies, that bind specifically to a polypeptide of theinvention. See, for instance, Wilson et al., Cell 37:767-778 (1984) at777.

Antigenic epitope-bearing peptides and polypeptides of the inventionpreferably contain a sequence of at least seven, more preferably atleast nine and most preferably between about 15 to about 30 amino acidscontained within the amino acid sequence of a polypeptide of theinvention. Non-limiting examples of antigenic polypeptides or peptidesthat can be used to generate FGF-13-specific antibodies include: apolypeptide comprising amino acid residues in SEQ ID NO:2 from about Gin22 to about Asn 32, about Asn 34 to about Met 43, about Gln 46 to aboutTyr 55, about Ser 59 to about Val 66, about Val 68 to about Phe 83,about Val 88 to about Glu 105, about Met 110 to about Val 128, about Phe153 to about His 173, about Leu 178 to about Gln 182, about Phe 185 toabout Gln 194, and about Val 198 to about Gln 213. These polypeptidefragments have been determined to bear antigenic epitopes of the FGF-13protein by the analysis of the Jameson-Wolf antigenic index, as shown inFIG. 3, above.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means. See, e.g., Houghten, R. A. (1985)“General method for the rapid solid-phase synthesis of large numbers ofpeptides: specificity of antigen-antibody interaction at the level ofindividual amino acids.” Proc. Natl. Acad. Sci. USA 82:5131-5135; this“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art. See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow, M. etal., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle, F. J. et al, J.Gen. Virol. 66:2347-2354 (1985). Immunogenic epitope-bearing peptides ofthe invention, i.e., those parts of a protein that elicit an antibodyresponse when the whole protein is the immunogen, are identifiedaccording to methods known in the art. See, for instance, Geysen et al.,supra. Further still, U.S. Pat. No. 5,194,392 to Geysen (1990) describesa general method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092 to Geysen (1989) describes a methodof detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) on PeralkylatedOligopeptide Mixtures discloses linear C1-C7-alkyl peralkylatedoligopeptides and sets and libraries of such peptides, as well asmethods for using such oligopeptide sets and libraries for determiningthe sequence of a peralkylated oligopeptide that preferentially binds toan acceptor molecule of interest. Thus, non-peptide analogs of theepitope-bearing peptides of the invention also can be made routinely bythese methods.

Fusion Proteins

As one of skill in the art will appreciate, FGF-13 polypeptides of thepresent invention and the epitope-bearing fragments thereof describedabove can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EP A 394,827; Traunecker et al., Nature 331:84-86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric FGF-13 protein orprotein fragment alone (Fountoulakis et al., J. Biochem. 270:3958-3964(1995)).

Antibodies

FGF-13-protein specific antibodies for use in the present invention canbe raised against the intact FGF-13 protein or an antigenic polypeptidefragment thereof, which may be presented together with a carrierprotein, such as an albumin, to an animal system (such as rabbit ormouse) or, if it is long enough (at least about 25 amino acids), withouta carrier.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody fragments (suchas, for example, Fab and F(ab′)2 fragments) which are capable ofspecifically binding to FGF-13 protein. Fab and F(ab′)2 fragments lackthe Fc fragment of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding of an intactantibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, thesefragments are preferred.

The antibodies of the present invention may be prepared by any of avariety of methods. For example, cells expressing the FGF-13 protein oran antigenic fragment thereof can be administered to an animal in orderto induce the production of sera containing polyclonal antibodies. In apreferred method, a preparation of FGF-13 protein is prepared andpurified to render it substantially free of natural contaminants. Such apreparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or FGF-13 protein binding fragments thereof).Such monoclonal antibodies can be prepared using hybridoma technology(Köhler et al., Nature 256:495 (1975); Köhler et al., Eur. J Immunol.6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976); Hammerlinget al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,(1981) pp. 563-681 ). In general, such procedures involve immunizing ananimal (preferably a mouse) with an FGF-13 protein antigen or, morepreferably, with an FGF-13 protein-expressing cell. Suitable cells canbe recognized by their capacity to bind anti-FGF-13 protein antibody.Such cells may be cultured in any suitable tissue culture medium;however, it is preferable to culture cells in Earle's modified Eagle'smedium supplemented with 10% fetal bovine serum (inactivated at about56° C.), and supplemented with about 10 g/l of nonessential amino acids,about 1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin. Thesplenocytes of such mice are extracted and fused with a suitable myelomacell line. Any suitable myeloma cell line may be employed in accordancewith the present invention; however, it is preferable to employ theparent myeloma cell line (SP20), available from the American TypeCulture Collection, Manassas, Va. After fusion, the resulting hybridomacells are selectively maintained in HAT medium, and then cloned bylimiting dilution as described by Wands et al. (Gastroenterology80:225-232 (1981)). The hybridoma cells obtained through such aselection are then assayed to identify clones which secrete antibodiescapable of binding the FGF-13 protein antigen.

Alternatively, additional antibodies capable of binding to the FGF-13protein antigen may be produced in a two-step procedure through the useof anti-idiotypic antibodies. Such a method makes use of the fact thatantibodies are themselves antigens, and that, therefore, it is possibleto obtain an antibody which binds to a second antibody. In accordancewith this method, FGF-13-protein specific antibodies are used toimmunize an animal, preferably a mouse. The splenocytes of such ananimal are then used to produce hybridoma cells, and the hybridoma cellsare screened to identify clones which produce an antibody whose abilityto bind to the FGF-13 protein-specific antibody can be blocked by theFGF-13 protein antigen. Such antibodies comprise anti-idiotypicantibodies to the FGF-13 protein-specific antibody and can be used toimmunize an animal to induce formation of further FGF-13protein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). Alternatively, FGF-13protein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

For in vivo use of anti-FGF-13 in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above Methods for producing chimericantibodies are known in the art. See, for review, Morrison, Science229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly et al.,U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al.,EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO 8702671;Boulianne et al., Nature 312:643 (1984); Neuberger et a., Nature 314:268(1985).

FGF-13-Related Disorders

Diagnosis

This invention is also related to the use of the genes of the presentinvention as part of a diagnostic assay for detecting diseases orsusceptibility to diseases related to the presence of mutations in thenucleic acid sequences encoding the polypeptide of the presentinvention.

Individuals carrying mutations in a gene of the present invention may bedetected at the DNA level by a variety of techniques. Nucleic acids fordiagnosis may be obtained from a patient's cells, such as from blood,urine, saliva, tissue biopsy and autopsy material. The genomic DNA maybe used directly for detection or may be amplified enzymatically byusing PCR (Saiki et al., Nature, 324: 163-166 (1986)) prior to analysis.RNA or cDNA may also be used for the same purpose. As an example, PCRprimers complementary to the nucleic acid encoding a polypeptide of thepresent invention can be used to identify and analyze mutations. Forexample, deletions and insertions can be detected by a change in size ofthe amplified product in comparison to the normal genotype. Pointmutations can be identified by hybridizing amplified DNA to radiolabeledRNA or alternatively, radiolabeled antisense DNA sequences. Perfectlymatched sequences can be distinguished from mismatched duplexes by RNaseA digestion or by differences in melting temperatures.

Genetic testing based on DNA sequence differences may be achieved bydetection of alteration in electrophoretic mobility of DNA fragments ingels with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 230:1242 (1985)).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,Restriction Fragment Length Polymorphisms (RFLP)) and Southern blottingof genomic DNA.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

The present invention also relates to a diagnostic assay for detectingaltered levels of FGF-13 proteins in various tissues since anover-expression of the proteins compared to normal control tissuesamples may detect the presence of abnormal cellular proliferation, forexample, a tumor. Assays used to detect levels of protein in a samplederived from a host are well-known to those of skill in the art andinclude radioimmunoassays, competitive-binding assays, Western Blotanalysis, ELISA assays and “sandwich” assay. An ELISA assay (Coligan, etal., Current Protocols in Immunology, 1(2), Chapter 6, (1991)) initiallycomprises preparing an antibody specific to an antigen to thepolypeptides of the present invention, preferably a monoclonal antibody.In addition a reporter antibody is prepared against the monoclonalantibody. To the reporter antibody is attached a detectable reagent suchas radioactivity, fluorescence or, in this example, a horseradishperoxidase enzyme. A sample is removed from a host and incubated on asolid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein like bovine serum albumen. Next,the monoclonal antibody is incubated in the dish during which time themonoclonal antibodies attach to any polypeptides of the presentinvention attached to the polystyrene dish. All unbound monoclonalantibody is washed out with buffer. The reporter antibody linked tohorseradish peroxidase is now placed in the dish resulting in binding ofthe reporter antibody to any monoclonal antibody bound to the protein ofinterest.

Unattached reporter antibody is then washed out. Peroxidase substratesare then added to the dish and the amount of color developed in a giventime period is a measurement of the amount of a polypeptide of thepresent invention present in a given volume of patient sample whencompared against a standard curve.

A competition assay may be employed wherein antibodies specific to apolypeptide of the present invention are attached to a solid support andlabeled FGF-13 and a sample derived from the host are passed over thesolid support and the amount of label detected, for example by liquidscintillation chromatography, can be correlated to a quantity of apolypeptide of the present invention in the sample. is A “sandwich”assay is similar to an ELISA assay. In a “sandwich” assay a polypeptideof the present invention is passed over a solid support and binds toantibody attached to a solid support. A second antibody is then bound tothe polypeptide of interest. A third antibody which is labeled andspecific to the second antibody is then passed over the solid supportand binds to the second antibody and an amount can then be quantified.

The present inventors have discovered that FGF-13 is expressed incancerous ovarian tissue as well as fetal kidney and fetal brain tissue.Thus, cancers of these tissues as well as other cancerous tissues inmammals can express significantly enhanced levels of the FGF-13 proteinand mRNA encoding the FGF-13 protein when compared to a corresponding“standard” level. Further, it is believed that enhanced levels of theFGF-13 protein can be detected in certain body fluids (e.g., sera,plasma, urine, and spinal fluid) from mammals with such a cancer whencompared to sera from mammals of the same species not having the cancer.Thus, the invention provides a diagnostic method useful during diagnosisof a disorder involving FGF-13 expression, including cancers, whichinvolves measuring the expression level of the gene encoding the FGF-13protein in ovarian, renal or neurological system tissue or other cellsor body fluid from an individual and comparing the measured geneexpression level with a standard FGF-13 gene expression level in thattissue, cell or fluid, whereby an increase or decrease in the geneexpression level compared to the standard is indicative of a disorderrelated to FGF-13 expression.

Where a diagnosis of a disorder in the ovarian, renal or neurologicalsystem, including diagnosis of a tumor, has already been made accordingto conventional methods, the present invention is useful as a prognosticindicator, whereby patients exhibiting enhanced or reduced FGF-13 geneexpression will experience a worse clinical outcome relative to patientsexpressing the gene at a level nearer the standard level.

By “assaying the expression level of the gene encoding the FGF-13protein” is intended qualitatively or quantitatively measuring orestimating the level of the FGF-13 protein or the level of the mRNAencoding the FGF-13 protein in a first biological sample either directly(e.g., by determining or estimating absolute protein level or mRNAlevel) or relatively (e.g., by comparing to the FGF-13 protein level ormRNA level in a second biological sample). Preferably, the FGF-13protein level or mRNA level in the first biological sample is measuredor estimated and compared to a standard FGF-13 protein level or mRNAlevel, the standard being taken from a second biological sample obtainedfrom an individual not having the disorder or being determined byaveraging levels from a population of individuals not having a disorderrelated to FGF-13 expression. As will be appreciated in the art, once astandard FGF-13 protein level or mRNA level is known, it can be usedrepeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, body fluid, cell line, tissue culture, or other sourcewhich contains FGF-13 protein or mRNA. As indicated, biological samplesinclude body fluids (such as sera, plasma, urine, synovial fluid andspinal fluid) which contain free FGF-13 protein, ovarian or renal systemtissue, and other tissue sources found to express complete or matureFGF-13 polypeptide or an FGF-13 receptor. Methods for obtaining tissuebiopsies and body fluids from mammals are well known in the art. Wherethe biological sample is to include mRNA, a tissue biopsy is thepreferred source.

Total cellular RNA can be isolated from a biological sample using anysuitable technique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding the FGF-13 protein are then assayed using any appropriatemethod. These include Northern blot analysis, S1 nuclease mapping, thepolymerase chain reaction (PCR), reverse transcription in combinationwith the polymerase chain reaction (RT-PCR), and reverse transcriptionin combination with the ligase chain reaction (RT-LCR).

Assaying FGF-13 protein levels in a biological sample can occur usingantibody-based techniques. For example, FGF-13 protein expression intissues can be studied with classical immunohistological methods(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-basedmethods useful for detecting FGF-13 protein gene expression includeimmunoassays, such as the enzyme linked immunosorbent assay (ELISA) andthe radioimmunoassay (RIA). Suitable antibody assay labels are known inthe art and include enzyme labels, such as, glucose oxidase, andradioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹²In), and technetium (^(99m)Tc), and fluorescentlabels, such as fluorescein and rhodamine, and biotin.

In addition to assaying FGF-13 protein levels in a biological sampleobtained from an individual, FGF-13 protein can also be detected in vivoby imaging. Antibody labels or markers for in vivo imaging of FGF-13protein include those detectable by X-radiography, NMR or ESR. ForX-radiography, suitable labels include radioisotopes such as barium orcesium, which emit detectable radiation but are not overtly harmful tothe subject. Suitable markers for NMR and ESR include those with adetectable characteristic spin, such as deuterium, which may beincorporated into the antibody by labeling of nutrients for the relevanthybridoma.

A FGF-13 protein-specific antibody or antibody fragment which has beenlabeled with an appropriate detectable imaging moiety, such as aradioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for immune systemdisorder. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of ^(99m)Tc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain FGF-13 protein. Invivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).

Treatment

The polypeptide of the present invention, as a result of the ability tostimulate vascular endothelial cell growth, may be employed in treatmentfor stimulating re-vascularization of ischemic tissues due to variousdisease conditions such as thrombosis, arteriosclerosis, and othercardiovascular conditions. These polypeptide may also be employed tostimulate angiogenesis and limb regeneration.

The polypeptide may also be employed for treating wounds due toinjuries, burns, post-operative tissue repair, and ulcers since they aremitogenic to various cells of different origins, such as fibroblastcells and skeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed stimulateneuronal growth and to treat and prevent neuronal damage associated withstroke and which occurs in certain neuronal disorders orneuro-degenerative conditions such as Alzheimer's disease, Parkinson'sdisease, and AIDS related complex. FGF-13 increases the number ofdopaminergic neurons surviving invitro indicating that it may benefitthe Parkinson's disease patient. FGF-13 has the ability to stimulatechondrocyte growth, therefore, they may be employed to enhance bone andperiodontal regeneration and aid in tissue transplants or bone grafts.

The polypeptide of the present invention may be also be employed toprevent skin aging due to sunburn by stimulating keratinocyte growth.

The FGF-13 polypeptide may also be employed for preventing hair loss,since FGF family members activate hair-forming cells and promotesmelanocyte growth. Along the same lines, the polypeptides of the presentinvention may be employed to stimulate growth and differentiation ofhematopoietic cells and bone marrow cells when used in combination withother cytokines.

The FGF-13 polypeptide may also be employed to maintain organs beforetransplantation or for supporting cell culture of primary tissues.

The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing such polypeptides, orpolynucleotides encoding such polypeptides, for in vitro purposesrelated to scientific research, synthesis of DNA, manufacture of DNAvectors and for the purpose of providing diagnostics and therapeuticsfor the treatment of human disease.

This invention provides a method for identification of the receptors forthe polypeptides of the present invention. The genes encoding thereceptor can be identified by numerous methods known to those of skillin the art, for example, ligand panning and FACS sorting (Coligan, etal., Current Protocols in Immun., 1(2), Chapter 5, (1991)). Preferably,expression cloning is employed wherein polyadenylated RNA is preparedfrom a cell responsive to the polypeptides, for example, NIH3T3 cellswhich are known to contain multiple receptors for the FGF familyproteins, and SC-3 cells, and a cDNA library created from this RNA isdivided into pools and used to transfect COS cells or other cells thatare not responsive to the polypeptides. Transfected cells which aregrown on glass slides are exposed to the polypeptide of the presentinvention, after they have been labeled. The polypeptides can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase.

Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

As a further approach to identifying the specific class(es) of knownFGF-specific receptors which bind FGF-13, recombinant human FGF-13protein was assayed for mitogenic activity on BaF3 cell lines engineeredto express individual FGF receptors (Ornitz et al., J. Biol. Chem.,271:15292-15297 (1996)). These experiments demonstrate that FGF-13preferentially activates FGF receptors 3c and 4 and has some activitytoward FGF receptors 1c and 2c. See FIG. 13. No activity was detectedtoward the “b” splice variants of any FGF receptor. Overall, this is asimilar receptor specificity pattern to that of FGF-8. However, theoverall activity of recombinant FGF-13 is considerably lower than thatof other FGFs, suggesting that the particular preparation of thisrecombinant protein which was tested was not fully active.

Formulations

The FGF-13 polypeptide composition will be formulated and dosed in afashion consistent with good medical practice, taking into account theclinical condition of the individual patient (especially the sideeffects of treatment with FGF-13 polypeptide alone), the site ofdelivery of the FGF-13 polypeptide composition, the method ofadministration, the scheduling of administration, and other factorsknown to practitioners. The “effective amount” of FGF-13 polypeptide forpurposes herein is thus determined by such considerations.

The polypeptides, agonists and antagonists of the present invention maybe employed in combination with a suitable pharmaceutical carrier tocomprise a pharmaceutical composition for parenteral administration.Such compositions comprise a therapeutically effective amount of thepolypeptide, agonist or antagonist and a pharmaceutically acceptablecarrier or excipient. Such a carrier includes but is not limited tosaline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. The formulation should suit the mode ofadministration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, thepolypeptides, agonists and antagonists of the present invention may beemployed in conjunction with other therapeutic compounds.

The pharmaceutical compositions may be administered in a convenientmanner such as by the oral, topical, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes. Thepharmaceutical compositions are administered in an amount which iseffective for treating and/or prophylaxis of the specific indication. Ingeneral, they are administered in an amount of at least about 10 μg/kgbody weight and in most cases they will be administered in an amount notin excess of about 8 mg/Kg body weight per day. In most cases, thedosage is from about 10 μg/kg to about 1 mg/kg body weight daily, takinginto account the routes of administration, symptoms, etc. In thespecific case of topical administration, dosages are preferablyadministered from about 0.1 μg to 9 mg per cm².

The polypeptide of the invention and agonist and antagonist compoundswhich are polypeptides, may also be employed in accordance with thepresent invention by expression of such polypeptide in vivo, which isoften referred to as “gene therapy.”

Thus, for example, cells may be engineered with a polynucleotide (DNA orRNA) encoding for the polypeptide ex vivo, the engineered cells are thenprovided to a patient to be treated with the polypeptide. Such methodsare well-known in the art. For example, cells may be engineered byprocedures known in the art by use of a retroviral particle containingRNA encoding for the polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expression of thepolypeptide in vivo, for example, by procedures known in the art. Asknown in the art, a producer cell for producing a retroviral particlecontaining RNA encoding the polypeptide of the present invention may beadministered to a patient for engineering cells in vivo and expressionof the polypeptide in vivo. These and other methods for administering apolypeptide of the present invention by such methods should be apparentto those skilled in the art from the teachings of the present invention.For example, the expression vehicle for engineering cells may be otherthan a retroviral particle, for example, an adenovirus, which may beused to engineer cells in vivo after combination with a suitabledelivery vehicle.

Retroviruses from which the retroviral plasmid vectors hereinabovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, spleen necrosis virus, retroviruses such as Rous SarcomaVirus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemiavirus, human immunodeficiency virus, adenovirus, .MyeloproliferativeSarcoma Virus, and mammary tumor virus. In one embodiment, theretroviral plasmid vector is derived from Moloney Murine Leukemia Virus.

The vector includes one or-more promoters. Suitable promoters which maybe employed include, but are not limited to, the retroviral LTR; theSV40 promoter; and the human cytomegalovirus (CMV) promoter described inMiller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or anyother promoter (e.g., cellular promoters such as eukaryotic cellularpromoters including, but not limited to, the histone, pol III, andβ-actin promoters). Other viral promoters which may be employed include,but are not limited to, adenovirus promoters, thymidine kinase (TK)promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

The nucleic acid sequence encoding the polypeptide of the presentinvention is under the control of a suitable promoter. Suitablepromoters which may be employed include, but are not limited to,adenoviral promoters, such as the adenoviral major late promoter; orhetorologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAl promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRshereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter also may be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, -2,-AM, PA12, T19-14X, VT-19-17-H2, CRE, CRIP, GP+E-86, GP+envAm12, and DANcell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14(1990), which is incorporated herein by reference in its entirety. Thevector may transduce the packaging cells through any means known in theart. Such means include, but are not limited to, electroporation, theuse of liposomes, and CaPO₄ precipitation. In one alternative, theretroviral plasmid vector may be encapsulated into a liposome, orcoupled to a lipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include the nucleic acid sequence(s) encoding the polypeptides.Such retroviral vector particles then may be employed, to transduceeukaryotic cells, either in vitro or in vivo. The transduced eukaryoticcells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

Agonists and Antagonists—Assays and Molecules

This invention provides a method of screening compounds to identifythose which modulate the action of the polypeptide of the presentinvention. An example of such an assay comprises combining a mammalianfibroblast cell, a the polypeptide of the present invention, thecompound to be screened and ³[H] thymidine under cell culture conditionswhere the fibroblast cell would normally proliferate. A control assaymay be performed in the absence of the compound to be screened andcompared to the amount of fibroblast proliferation in the presence ofthe compound to determine if the compound stimulates proliferation bydetermining the uptake of ³[H] thymidine in each case. The amount offibroblast cell proliferation is measured by liquid scintillationchromatography which measures the incorporation of ³[H] thymidine. Bothagonist and antagonist compounds may be identified by this procedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for a polypeptide of the present invention (as described aboveand in (Ornitz et al., supra) is incubated with a labeled polypeptide ofthe present invention in the presence of the compound. The ability ofthe compound to enhance or block this interaction could then bemeasured. Alternatively, the response of a known second messenger systemfollowing interaction of a compound to be screened and the FGF-13receptor is measured and the ability of the compound to bind to thereceptor and elicit a second messenger response is measured to determineif the compound is a potential agonist or antagonist. Such secondmessenger systems include but are not limited to, cAMP guanylatecyclase, ion channels or phosphoinositide hydrolysis.

Examples of antagonist compounds include antibodies, or in some cases,oligonucleotides, which bind to the receptor for the polypeptide of thepresent invention but elicit no second messenger response or bind to theFGF-13 polypeptide itself. Alternatively, a potential antagonist may bea mutant form of the polypeptide which binds to the receptors, however,no second messenger response is elicited and, therefore, the action ofthe polypeptide is effectively blocked.

Another antagonist compound to the FGF-13 gene and gene product is anantisense construct prepared using antisense technology. Antisensetechnology can be used to control gene expression through triple-helixformation or antisense DNA or RNA, both of which methods are based onbinding of a polynucleotide to DNA or RNA. For example, the 5′ codingportion of the polynucleotide sequence, which encodes for the maturepolypeptides of the present invention, is used to design an antisenseRNA oligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervanet al., Science, 251: 1360 (1991)), thereby preventing transcription andthe production of the polypeptides of the present invention. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into the polypeptide (Antisense—Okano,J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as AntisenseInhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)). Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of the polypeptide.

Potential antagonist compounds also include small molecules which bindto and occupy the binding site of the receptors thereby making thereceptor inaccessible to its polypeptide such that normal biologicalactivity is prevented. Examples of small molecules include, but are notlimited to, small peptides or peptide-like molecules.

Antagonist compounds may be employed to inhibit the cell growth andproliferation effects of the polypeptides of the present invention onneoplastic cells and tissues, i.e. stimulation of angiogenesis oftumors, and, therefore, retard or prevent abnormal cellular growth andproliferation, for example, in tumor formation or growth.

The antagonists may also be employed to prevent hyper-vascular diseases,and prevent the proliferation of epithelial lens cells afterextracapsular cataract surgery. Prevention of the mitogenic activity ofthe polypeptides of the present invention may also be desirous in casessuch as restenosis after balloon angioplasty.

The antagonists may also be employed to prevent the growth of scartissue during wound healing.

Chromosome Assays

In certain preferred embodiments relating to chromosomal mapping, thecDNA herein disclosed-is used to clone genomic DNA of an FGF-13 gene.This can be accomplished using a variety of well known techniques andlibraries, which generally are available commercially. The genomic DNAthen is used for in situ chromosome mapping using well known techniquesfor this purpose. Therefore, the nucleic acid molecules of the presentinvention are also valuable for chromosome identification. The sequenceis specifically targeted to and can hybridize with a particular locationon an individual human chromosome. Moreover, there is a current need foridentifying particular sites on the chromosome. Few chromosome markingreagents based on actual sequence data (repeat polymorphisms) arepresently available for marking chromosomal location. The mapping ofDNAs to chromosomes according to the present invention is an importantfirst step in correlating those sequences with genes associated withdisease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3′untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, thus complicating theamplification process. These primers are then used for PCR screening ofsomatic cell hybrids containing individual human chromosomes. Only thosehybrids containing the human gene corresponding to the primer will yieldan amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that cansimilarly be used to map to its chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNA as short as 50 or 60bases. For a review of this technique, see Verma et al., HumanChromosomes: a Manual of Basic Techniques, Pergamon Press, New York(1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man (available on line through JohnsHopkins University Welch Medical Library). The relationship betweengenes and diseases that have been mapped to the same chromosomal regionare then identified through linkage analysis (coinheritance ofphysically adjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes. (This assumes 1 megabase mapping resolution and onegene per 20 kb).

Using methods described above, the FGF-13 gene of the invention has beenmapped by florescent in situ hybridization to human chromosome 8p21. Thecorresponding map position in the mouse includes several disease lociincluding ds (disorganization—developmental disruption) and wc (wavedcoat—homozygous lethality).

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES

The present invention will be further described with reference to thefollowing examples; however, it is to be understood that the presentinvention is not limited to such examples. All parts or amounts, unlessotherwise specified, are by weight.

In order to facilitate understanding of the following examples, certainfrequently occurring methods and/or terms will be described.

“Plasmids” are designated by a lower case p. preceded and/or followed bycapital letters and/or numbers. The starting plasmids herein are eithercommercially available, publicly available on an unrestricted basis, orcan be constructed from available plasmids in accord with publishedprocedures. In addition, equivalent plasmids to those described areknown in the art and will be apparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion the reaction is electrophoreseddirectly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percentpolyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.,8:4057 (1980).

Oligonucleotides” refers to either a single stranded polydeoxynucleotideor two complementary polydeoxynucleotide strands which may be chemicallysynthesized. Such synthetic oligonucleotides have no 5′ phosphate andthus will not ligate to another oligonucleotide without adding aphosphate with an ATP in the presence of a kinase. A syntheticoligonucleotide will ligate to a fragment that has not beendephosphorylated.

“Ligation” refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (Maniatis, T., et al., Id.,p. 146). Unless otherwise provided, ligation may be accomplished usingknown buffers and conditions with 10 units of T4 DNA ligase (“ligase”)per 0.5 μg of approximately equimolar amounts of the DNA fragments to beligated.

Unless otherwise stated, transformation was performed as described bythe method of Graham, F. and Van der Eb, A., Virology, 52:456-457(1973).

Example 1(a) Expression and Purification of “His-tagged” FGF-13 in E.coli

The DNA sequence encoding FGF-13 ATCC # 97148, was initially amplifiedusing PCR oligonucleotide primers corresponding to the 5′ sequences ofthe polypeptide having the amino acid sequence from position 2 toposition 193 of SEQ ID NO:2 and to the vector sequences 3′ to the gene.Additional nucleotides corresponding to the gene were added to the 5′and 3′ sequences respectively. The 5′ oligonucleotide primer 5′GCCAGACCATGGAGAATCACCCGTCTCCTAAT 3′ (SEQ ID NO:11) contains a Ncorestriction enzyme site. The 3′ sequence 5′GATTTAAGATCTCGTGAGGGGCTGGGGCCG 3′ (SEQ ID NO:12) contains complementarysequences to a BglII site and is followed by 18 nucleotides of FGF-13coding sequence.

The restriction enzyme sites correspond to the restriction enzyme siteson the bacterial expression vector pQE-60 (Qiagen, Inc. Chatsworth,Calif. 91311). pQE-60 encodes antibiotic resistance (Amp^(r)), abacterial origin of replication (ori), an IPTG-regulatable promoteroperator (P/O), a ribosome binding site (RBS), a 6-His tag andrestriction enzyme sites. pQE-60 was then digested with NcoI and BglII.The amplified sequences were ligated into pQE-60 and were inserted inframe with the sequence encoding for the histidine tag and the ribosomebinding site (RBS). The ligation mixture was then used to transform E.coli strain M15/rep 4 (Qiagen, Inc.) by the procedure described inSambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold SpringLaboratory Press, (1989). M15/rep4 contains multiple copies of theplasmid pREP4, which expresses the lacI repressor and also conferskanamycin resistance (Kan^(r)). Transformants were identified by theirability to grow on LB plates and ampicillin/kanamycin resistant colonieswere selected. Plasmid DNA was isolated and confirmed by restrictionanalysis.

Clones containing the desired constructs are grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells are grown to an optical density 600(O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG (“Isopropyl-B-D-thiogalactopyranoside”) is then added to a final concentration of 1 mM. IPTGinduces by inactivating the lacI repressor, clearing the P/O leading toincreased gene expression. Cells are grown an extra 3 to 4 hours. Cellsare then harvested by centrifugation. The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl. After clarification,solubilized FGF-13 is purified from this solution by chromatography on aNickel-Chelate column under conditions that allow for tight binding byproteins containing the 6-His tag (Hochuli, E. et al., J. Chromatography411:177-184 (1984)). The proteins are eluted from the column in 6 molarguanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3molar guanidine HCl, 100 mM sodium phosphate, 10 mmolar glutathione(reduced) and 2 mmolar glutathione (oxidized). After incubation in thissolution for 12 hours the proteins are dialyzed to 10 mmolar sodiumphosphate.

Example 1(b) Expression and Purification of FGF-13 in E. coli

The bacterial expression vector pQE70 is used for bacterial expressionin this example (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,91311). pQE70 encodes ampicillin antibiotic resistance (“Amp”) andcontains a bacterial origin of replication (“ori”), an IPTG induciblepromoter, a ribosome binding site (“RBS”), six codons encoding histidineresidues that allow affinity purification usingnickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN,Inc., supra, and suitable single restriction enzyme cleavage sites.These elements are arranged such that a DNA fragment encoding apolypeptide may be inserted in such as way as to produce thatpolypeptide with the six His residues (i.e., a “6×His tag”) covalentlylinked to the carboxyl terminus of that polypeptide. However, in thisexample, the polypeptide coding sequence is inserted such thattranslation of the six His codons is prevented and, therefore, thepolypeptide is produced with no 6×His tag.

The DNA sequence encoding the desired portion of the FGF-13 proteincomprising the predicted mature form of the FGF-13 amino acid sequence(i.e., amino acids 1-193 of SEQ ID NO:2) was amplified from thedeposited cDNA clone using PCR oligonucleotide primers which anneal tothe amino terminal sequences encoding the desired portion of the FGF-13protein and to sequences in the deposited construct 3′ to the cDNAcoding sequence. Additional nucleotides containing restriction sites tofacilitate cloning in the pQE70 vector were added to the 5′ and 3′sequences, respectively.

For cloning mature form of the FGF-13 protein, the 5′ primer had thesequence 5′ CTAGTCGCATGCAGGGGGAGAATCACCCGTCT 3′ (SEQ ID NO:13)containing the underlined SphI restriction site, which includes aninitiation codon and following the initiation codon, 21 nucleotides ofthe amino terminal coding sequence of the mature FGF-13 sequence in SEQID NO:2. One of ordinary skill in the art would appreciate, of course,that the point in the protein coding sequence where the 5′ primer beginsmay be varied to amplify a desired portion of the complete proteinshorter or longer than the mature form. The 3′ primer had the sequence5′ GCTTGAAAGCTTCTACGTGAGGGGCTGGGGCCG 3′ (SEQ ID NO:14) containing theunderlined HindIII restriction site followed by a stop codon and 18nucleotides complementary to the 3′ end of the coding sequence in theFGF-13 DNA sequence in SEQ ID NO:1.

The amplified FGF-13 DNA fragments and the vector pQE70 were digestedwith SphI and HindIII and the digested DNAs were then ligated together.Insertion of the FGF-13 DNA into the restricted pQE70 vector places theFGF-13 protein coding region including its associated stop codondownstream from the IPTG-inducible promoter and in-frame with theinitiating AUG in the 5′ primer. The associated stop codon preventstranslation of the six histidine codons downstream of the insertionpoint.

The ligation mixture was transformed into competent E. coli cells usingstandard procedures such as those described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance(“Kan^(r)”), was used in carrying out the illustrative example describedherein. This strain, which is only one of many that are suitable forexpressing FGF-13 protein, is available commercially from QIAGEN, Inc.,supra. Transformants were identified by their ability to grow on LBplates in the presence of ampicillin and kanamycin. Plasmid DNA wasisolated from resistant colonies and the identity of the cloned DNAconfirmed by restriction analysis, PCR and DNA sequencing.

Clones containing the desired constructs were grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml). The O/N culture was used to inoculate a largeculture, at a dilution of approximately 1:25 to 1:250. The cells aregrown to an optical density at 600 nm (“OD⁶⁰⁰”) of between 0.4 and 0.6.Isopropyl-b-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacI repressor. Cellssubsequently were incubated further for 3 to 4 hours and were thenharvested by centrifugation.

To purify the FGF-13 polypeptide, the cells were lysed in amicrofluidizer and then stirred for 3-4 hours at 4° C. in 2M and then 6Mguanidine-HCl, 50 mM tris-HCl, pH 7.5, 2 mM EDTA. FGF-13 protein waspresent in both the 2M and 6M GuHCl extracts. The combined GuHCl extractwas quickly diluted into a buffer containing 30 mM Tris pH7.5, 5 mMEDTA, 200 mM NaCl, 20 ug/ml Pefabloc SC, 2 ug/ml E-64 (BoeringerMannhein). The refolded FGF-13 was purified through poros 50 HS(PerSeptive Biosystem) cation exchange column at pH7. The HS purifiedprotein was applied to a set of poros HQ 50/poros CM 20 (PerSeptiveBiosystem) anion/cation columns in a tandem chromatographic mode. FGF-13was eluted from the CM column with 20 column volumes of 0.2 to 1.25MNaCl linear gradient in 24 mM NaCl, and purification was finished with aS200 sepharcryl HR (Pharmacia) size exclusion column.

The GuHCl extracted protein appeared to be the same size as the startingmaterial on SDS-PAGE and was greater than 60% pure. However, afterrefolding the protein showed three bands which appeared to be about 2 kDsmaller on SDS-PAGE, suggesting that proteolytic degradation may haveoccurred during refolding. Refolded FGF-13 captured by a strong cationexchange on the poros HS 50 column eluted at 80% purity with 1M NaCl.The protein resulting from the set of tandem columns, which was elutedfrom the CM column with 600 mM NaCl, showed at least three differentbands on SDS-PAGE: two upper bands at about 22 kD and one lower band atabout 19 kD. In an attempt to separate the upper and lower bands, the CMpurified FGF-13 was put through a S200 sepharcryl HR size exclusioncolumn. A fraction containing mainly the upper bands was isolated. Theupper bands and the lower band were analyzed by N-terminusmicrosequencing.

The purified FGF-13 was slot blotted onto a ProBlott membrane (AppliedBiosystems, Inc. (ABI) and stained with Ponceau S (0.2% in 3% aceticacid). The band of interest was then excised, placed in a “BlotCartridge” and subjected to N-terminal amino acid sequence analysisusing a model ABI-494 sequencer (Perkin-Elmer-Applied Biosystems, Inc.)and the Gas-phase Blot cycles. The results showed that the N-terminalsequence of the upper doublet bands was as predicted for the QE70expression construct including the N-terminal Met (i.e., MQGEN . . . )while the lower MW band had an N-terminal sequence 21 amino acidsshorter (i.e., TDQLS). These terminal sequences represented,respectively, 40% and 50% of all N-termini in the originalunfractionated preparation. Fibroblast proliferation assays showed thatthe upper and lower fractions exhibited comparable, and both fractionswere more active than the His-tagged FGF-13 made using the vector ofExample 1(a). These assays also showed that FGF-13 can be frozen at −80°C. and thawed later without losing its activity. The upper and lowerFGF-13 fractions were then combined for all further biological activitytests described herein, producing a mixture consisting of three bandswith nearly equal intensity on SDS-PAGE, greater than 95% purity and alow endotoxin level of 2 EU/mg.

Example 2 Cloning and Expression of FGF-13 Protein in a BaculovirusExpression System

The DNA sequence encoding the full length FGF-13 protein, ATCC #97148,is amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ sequences of the gene:

The FGF-13 5′ primer has the sequence 5′ CTAGTGGATCCCGA

GAATCACCCGTCTCCT 3′ (SEQ ID NO:15) and contains a BamHI restrictionenzyme site (in bold) such that cloning at this site will put thebaculovirus signal sequence in frame with 18 nucleotides of the FGF-13gene downstream of the putative FGF-13 signal peptide cleavage site.

The 3′ primer has the sequence 5′ CGACTTCTAGAACCT

CGGGGATCTGGCTCC 3′ (SEQ ID NO:16) and contains the cleavage site for therestriction endonuclease XbaI and 18 nucleotides complementary to the 3′non-translated sequence of the gene. The amplified sequences areisolated from a 1% agarose gel using a commercially available kit(“Geneclean,” BIO 101 Inc., La Jolla, Calif.). The fragment is thendigested with the respective endonucleases and purified again on a 1%agarose gel. This fragment is designated F2.

The vector pA2gp (modification of pVL941 vector, discussed below) isused for the expression of the proteins using the baculovirus expressionsystem (for review see: Summers, M. D. and Smith, G. E. 1987, A manualof methods for baculovirus vectors and insect cell culture procedures,Texas Agricultural Experimental Station Bulletin No. 1555). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases BamHI and XbaI.The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusthe beta-galactosidase gene from E.coli is inserted in the sameorientation as the polyhedrin promoter followed by the polyadenylationsignal of the polyhedrin gene. The polyhedrin sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of co-transfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pA2 such as pRG1, pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid is digested with the restriction enzymes anddephosphorylated using calf intestinal phosphatase. by procedures knownin the art. The DNA is then isolated from a 1% agarose gel using thecommercially available kit (“Geneclean” BIO 101 Inc., La Jolla, Calif.).This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNAligase. E.coli DH5 cells are then transformed and bacteria identifiedthat contained the plasmid (pBacFGF-13) using the respective restrictionenzymes. The sequence of the cloned fragment are confirmed by DNAsequencing.

5 μg of the plasmid pBacFGF-13 are co-transfected with 1.0 μg of acommercially available linearized baculovirus (“BaculoGold™ baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 μg of BaculoGold™ virus DNA and 5 μg of the plasmids, in each case,are mixed in a sterile well of microtiter plates containing 50 μl ofserum free Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture is added drop-wise to the Sf9 insect cells (ATCC CRL 1711)seeded in 35 mm tissue culture plates with 1 ml Grace's medium withoutserum. The plates are rocked back and forth to mix the newly addedsolution. The plates are then incubated for 5 hours at 27%C. After 5hours the transfection solution is removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum is added.The plates are put back into an incubator and cultivation continued at27%C for four days.

After four days the supernatant is collected and plaque assays performedsimilar as described by Summers and Smith (supra). As a modification anagarose gel with “Blue Gal” (Life Technologies Inc., Gaithersburg) isused which allows an easy isolation of blue stained plaques. (A detaileddescription of a “plaque assay” can also be found in the user's guidefor insect cell culture and baculovirology distributed by LifeTechnologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution the virus is added to the cells andblue stained plaques are picked with the tip of an Eppendorf pipette.The agar containing the recombinant viruses is then resuspended in anEppendorf tube containing 200 μl of Grace's medium. The agar is removedby a brief centrifugation and the supernatant containing the recombinantbaculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Fourdays later the supernatants of these culture dishes are harvested andthen stored at 4%C.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-FGF-13 at a multiplicity of infection (MOI) of 2. Sixhours later the medium is removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 μCi of 35S-methionine and 5 μCi 35S cysteine (Amersham)are added. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labelled proteins visualized bySDS-PAGE and autoradiography.

Example 3 Cloning and Expression of FGF-13 in Mammalian Cells Example3(a) Cloning and Expression in COS Cells

The expression of the predicted mature FGF-13 polypeptide uses aplasmid, FGF-13-HA derived from a vector pcDNA3/Amp (Invitrogen)containing: 1) SV40 origin of replication, 2) ampicillin resistancegene, 3) E.coli replication origin, 4) CMV promoter followed by apolylinker region, an SV40 intron and polyadenylation site. DNAfragments encoding the entire FGF-13 precursor and an HA tag fused inframe to the 3′ end is cloned into the polylinker region of the vector,therefore, the recombinant protein expression is directed under the CMVpromoter. The HA tag corresponds to an epitope derived from theinfluenza hemagglutinin protein as previously described (I. Wilson, H.Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell37:767, (1984)). The infusion of HA tag to the target protein allowseasy detection of the recombinant protein with an antibody thatrecognizes the HA epitope.

The PCR amplified DNA fragments and the vector, pcDNA3/Amp, are digestedwith the respective restriction enzymes and ligated. The ligationmixture is transformed into E. coli strain SURE (Stratagene CloningSystems, La Jolla, Calif.) the transformed culture is plated onampicillin media plates and resistant colonies are selected. Plasmid DNAis isolated from transformants and examined by restriction analysis forthe presence of the correct fragment. For expression of the recombinantFGF-13 COS cells are transfected with the expression vector byDEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, MolecularCloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)). Theexpression of the FGF-13-HA protein is detected by radiolabelling andimmunoprecipitation method (E. Harlow, D. Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelledfor 8 hours with 35S-cysteine two days post transfection. Culture mediais then collected and cells are lysed with detergent (RIPA buffer (150mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM Tris, pH 7.5)(Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culturemedia are precipitated with an HA specific monoclonal antibody. Proteinsprecipitated are analyzed on 15% SDS-PAGE gels.

Example 3(b) Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of FGF-13 polypeptide. PlasmidpC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146).To produce a soluble, secreted form of the polypeptide, the predictedmature is fused to the secretory leader sequence of the human IL-6 gene.The plasmid contains the mouse DHFR gene under control of the SV40 earlypromoter. Chinese hamster ovary- or other cells lacking dihydrofolateactivity that are transfected with these plasmids can be selected bygrowing the cells in a selective medium (alpha minus MEM, LifeTechnologies) supplemented with the chemotherapeutic agent methotrexate.The amplification of the DHFR genes in cells resistant to methotrexate(MTX) has been well documented (see, e.g., Alt, F. W., Kellems, R. M.,Bertino, J. R., and Schimke; R. T., 1978, J. Biol. Chem. 253:1357-1370,Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta, 1097:107-143,Page, M. J. and Sydenham, M. A. 1991, Biotechnology 9:64-68). Cellsgrown in increasing concentrations of MTX develop resistance to the drugby overproducing the target enzyme, DHFR, as a result of amplificationof the DHFR gene. If a second gene is linked to the DHFR gene, it isusually co-amplified and over-expressed. It is known in the art thatthis approach may be used to develop cell lines carrying more than 1,000copies of the amplified gene(s). Subsequently, when the methotrexate iswithdrawn, cell lines are obtained which contain the amplified geneintegrated into one or more chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rouse Sarcoma Virus(Cullen, et al., Molecular and Cellular Biology, March 1985:438-447)plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530 (1985)).Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: BamHI, Xba I,and Asp718. Behind these cloning sites the plasmid contains the 3′intron and polyadenylation site of the rat preproinsulin gene. Otherhigh efficiency promoters can also be used for the expression, e.g., thehuman β-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the FGF-13 polypeptide in a regulated wayin mammalian cells (Gossen, M., & Bujard, H. 1992, Proc. Natl. Acad.Sci. USA 89:5547-5551). For the polyadenylation of the mRNA othersignals, e.g., from the human growth hormone or globin genes can be usedas well. Stable cell lines carrying a gene of interest integrated intothe chromosomes can also be selected upon co-transfection with aselectable marker such as gpt, G418 or hygromycin. It is advantageous touse more than one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC4 is digested with the restriction enzymes BamHI and XbaIand then dephosphorylated using calf intestinal phosphates by proceduresknown in the art. The vector is then isolated from a 1% agarose gel.

The DNA sequence encoding the mature FGF-13 polypeptide is amplifiedusing PCR oligonucleotide primers corresponding to the 5′ and 3′sequences of the desired portion of the gene. The 5′ primer containingthe underlined BamHI site which overlaps with a Kozak sequence, an AUGstart codon, a sequence encoding the secretory leader peptide from thehuman IL-6 gene, and 21 nucleotides of the 5′ coding region of themature FGF-13 polypeptide, has the following sequence: 5′CTAGCCGGATCCGCCACCATGAACTCCTTCTCCACAAGCGCCTTCGGTCCAGTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTGCCCCAGTTGTGAGACCAGGGGGAGAATCACCCGTCT3′ (SEQ ID NO:17). The 3′ primer, containing the underlined XbaI and 18of nucleotides complementary to the 3′ coding sequence immediatelybefore the stop codon as shown in FIG. 1 (SEQ ID NO:1), has thefollowing sequence: 5′ GCTTGATCTAGACGTGAGGGGCTGGGGCCG 3′ (SEQ ID NO:18).

The amplified fragment is digested with the endonucleases BamHI and XbaIand then purified again on a 1% agarose gel. The isolated fragment andthe dephosphorylated vector are then ligated with T4 DNA ligase. E. coliHB101 or XL-1 Blue cells are then transformed and bacteria areidentified that contain the fragment inserted into plasmid pC4 using,for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. Five μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSVneo using lipofectin (Felgner et al.,supra). The plasmid pSV2-neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 4 Expression via Gene Therapy

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in tissue-culture medium and separated into smallpieces. Small chunks of the tissue are placed on a wet surface of atissue culture flask, approximately ten pieces are placed in each flask.The flask is turned upside down, closed tight and left at roomtemperature over night. After 24 hours at room temperature, the flask isinverted and the chunks of tissue remain fixed to the bottom of theflask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillinand streptomycin, is added. This is then incubated at 37%C forapproximately one week. At this time, fresh media is added andsubsequently changed every several days. After an additional two weeksin culture, a monolayer of fibroblasts emerge. The monolayer istrypsinized and scaled into larger flasks.

pMV-7 (Kirschmeier, P. T. et al, DNA, 7:219-25 (1988) flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding a polypeptide of the present invention is amplifiedusing PCR primers which correspond to the 5′ and 3′ end sequencesrespectively. The 5′ primer containing an EcoRI site and the 3′ primerhaving contains a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the EcoRI and HindIII fragment areadded together, in the presence of T4 DNA ligase. The resulting mixtureis maintained under conditions appropriate for ligation of the twofragments. The ligation mixture is used to transform bacteria HB 101,which are then plated onto agar-containing kanamycin for the purpose ofconfirming that the vector had the gene of interest properly inserted.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the gene is then added to the media and the packaging cellsare transduced with the vector. The packaging cells now produceinfectious viral particles containing the gene (the packaging cells arenow referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product.

Example 5 FGF-13 Biological Effects

Astrocyte and Neuronal Assays

Recombinant FGF-13, expressed in Escherichia coli and purified asdescribed above, was tested for activity in promoting the survival,neuritic outgrowth, or phenotypic differentiation of cortical neuronalcells and for inducing the proliferation of glial fibrillary acidicprotein immunopositive cells, astrocytes. The selection of corticalcells for the bioassay is based on the prevalent expression of FGF-1 andFGF-2 in cortical structures and on the previously reported enhancementof cortical neuronal survival resulting from FGF-2 treatment.

Based on absorption measurements made with calcein AM, treatment withFGF-13 produced a dose dependent increase in the number of cells incortical cultures (FIG. 4). Half-maximal and saturating responses toFGF-13 were observed at approximately 10 and 50 ng/ml, respectively, andwere nearly equivalent, at saturation, to those observed with FGF-2, apreviously characterized trophic factor for cortical neurons.

Since a change in calcein AM absorption does not discriminate between anincrease in the glial or neuronal cell compartment, FGF-13 was tested tosee whether it would induce a change in the level of phenotypicdifferentiation of one of the neuronal populations, the GABAergicneurons, present in the cortical cultures. After a 7 day treatmentperiod, the level of high-affinity GABA-uptake increased as a functionof the concentration of FGF-13 (FIG. 5). The GABAergic neuronal responseappeared to be more sensitive to FGF-13 than the general cell survivalresponse since the maximal induction of GABA-uptake occurred with 10 asopposed to 50 ng/ml of FGF-13.

Previous reports describing the biological effects of FGF-2 (basic FGF)on cortical or hippocampal neurons in vitro have demonstrated increasesin both neuron survival and neurite outgrowth (Walicke, P. et al.,“Fibroblast growth factor promotes survival of dissociated hippocampalneurons and enhances neurite extension.” Proc. Natl. Acad. Sci. USA83:3012-3016. (1986)). However, reports from experiments done on PC-12cells suggest that these two responses are not necessarily synonymousand may depend on not only which FGF is being tested but also on whichreceptor(s) are expressed on the target cells. Using the primarycortical neuronal culture paradigm, the ability of FGF-13 to induceneurite outgrowth was compared to the response achieved with FGF-2 (FIG.6). Saturating responses to FGF-2 and FGF-13 were achieved with 10 and50 ng/ml, respectively.

Astrocytes are the major non-neuronal cell type present in the corticalcultures. FGF-1 (acidic FGF), FGF-2, and FGF-9 are mitogens forastrocytes. As shown in FIG. 7, FGF-1, FGF-2, or FGF-9 produced a 5- to10-fold increase in the level of [³H]-thymidine incorporation. Ingeneral, maximal responses were achieved in the range of 10 to 50 ng/ml.In comparison, treatment with FGF-13, in the presence of 100 ng/mlheparin, produced a concentration dependent increase in [³ H]-thymidineincorporation up to 150 ng/ml. The addition of heparin caused anapparent shift to the left in the dose response curve.

Heterologous ligand competition binding studies were conducted onmembranes prepared from adult rat cortex. In these studies, the abilityof FGF-13 to displace [¹²⁵I]-FGF-1 was monitored and compared to thedisplacement achieved with FGF-2. FIG. 8 summarizes the displacementcurves for FGF-2, FGF-10, and FGF-13. The concentration of FGF-2 orFGF-13 required to achieve 50% displacement was 200 and 1000 pM,respectively.

Fibroblast and Endothelial Cell Assays

Human lung fibroblasts were obtained from Clonetics (San Diego, Calif.)and maintained in growth media from Clonetics. Dermal microvascularendothelial cells were obtained from Cell Applications (San Diego,Calif.). For proliferation assays, the human lung fibroblasts and dermalmicrovascular endothelial cells were cultured at 5,000 cells/well in a96-well plate for one day in growth medium. The cells were thenincubated for one day in 0.1% BSA basal medium. After replacing themedium with fresh 0.1% BSA medium, the cells were incubated with thetest proteins for 3 days. Alamar Blue (Alamar Biosciences, Sacramento,Calif.) was added to each well to a final concentration of 10%. Thecells were incubated for 4 hr. Cell viability was measured by reading ina CytoFluor fluorescence reader. For the PGE₂ assays, the human lungfibroblasts were cultured at 5,000 cells/well in a 96-well plate for oneday. After a medium change to 0.1% BSA basal medium, the cells wereincubated with FGF-2 or FGF-13 with or without IL-1α for 24 hours. Thesupernatants were collected and assayed for PGE₂ by EIA kit (Cayman, AnnArbor, Mich.). For the IL-6 assays, the human lung fibroblasts werecultured at 5,000 cells/well in a 96-well plate for one day. After amedium change to 0.1% BSA basal medium, the cells were incubated withFGF-2 or FGF-13 with or without IL-1α for 24 hours. The supernatantswere collected and assayed for IL-6 by ELISA kit (Endogen, Cambridge,Mass.).

Human lung fibroblasts were cultured with FGF-2 or FGF-13 for 3 days inbasal medium before the addition of Alamar Blue to assess effects ongrowth of the fibroblasts. FGF-2 showed stimulation at 10-2500 ng/mlwhile FGF-13 showed stimulation at 1000-2500 ng/ml (FIG. 9). However,the maximal effect was similar. In contrast to FGF-2, FGF-13 did nothave any stimulatory effect on dermal endothelial cells (FIG. 10).

FGF-2 and FGF-13 did not have any effect on PGE₂ and IL-6 release fromthe fibroblasts. IL-1α showed stimulation of PGE₂ and IL-6. Both FGF-2and FGF-13 acted synergistically with IL-1α to release PGE₂ (FIG. 11)and IL-6 (FIG. 12). FGF-13 at 2,500 ng/ml gave a similar effects as 100ng/ml FGF-2. Indomethacin at 100 ng/ml inhibited PGE₂ release but notIL-6 release from the fibroblasts.

Angiogenesis Assays

In vivo angiogenesis assay of FGF-13 measures the ability of an existingcapillary network to form new vessels in an implanted capsule of murineextracellular matrix material (Matrigel). The protein is mixed with theliquid Matrigel at 4° C. and the mixture is then injected subcutaneouslyin mice where it solidifies. After 7 days, the solid “plug” of Matrigelis removed and examined for the presence of new blood vessels. Matrigelwas purchased from Becton Dickinson Labware/Collaborative BiomedicalProducts.

When thawed at 4° C. the Matrigel material is a liquid. The Matrigel wasmixed with FGF-13 at 150 ng/ml at 4° C. and drawn into cold 3 mlsyringes. Female C57B1/6 mice approximately 8 weeks old were injectedwith the mixture of Matrigel and experimental protein at 2 sites at themidventral aspect of the abdomen (0.5 ml/site). After 7 days, the micewere sacrificed by cervical dislocation, the Matrigel plugs were removedand cleaned (i.e., all clinging membranes and fibrous tissue isremoved). Replicate whole plugs were fixed in neutral buffered 10%formaldehyde, embedded in paraffin and used to produce sections forhistological examination after staining with Masson's Trichrome. Crosssections from 3 different regions of each plug were processed. Selectedsections were stained for the presence of vWF. The positive control forthis assay was bovine basic FGF. (150 ng/ml). Matrigel alone was used todetermine basal levels of angiogenesis.

FGF-13 was weakly positive in that a small number of infiltrating ofcells were observed at the peripheral edge of the Matrigel plug.Immunostaining with antibody to vWF did not reveal vWF-positiveendothelial cells. Protocols such as the above which are known in theart generally quantify angiogenesis only by determining the totalcellularity of the plug, a procedure which may not give a completeassessment of angiogenic activity of the protein.

Parkinson's Model

The observed loss of motor function in Parkinson's disease is attributedto a deficiency of striatal dopamine resulting from the degeneration ofthe nigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine.(MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP+) and released.Subsequently, MPP⁺ is actively accumulated in dopaminergic neurons bythe high-affinity reuptake transporter for dopamine. MPP⁺ is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basicFGF) has trophic activity towards nigral dopaminergic neurons (Ferrariet al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

Based on the data with FGF-2, FGF-13 was evaluated to determine whetherit has an action similar to that of FGF-2 in enhancing dopaminergicneuronal survival in vitro and it was then be tested in vivo forprotection of dopaminergic neurons in the striatum from the damageassociated with MPTP treatment. The potential effect of FGF-13 was firstexamined in vitro in a dopaminergic neuronal cell culture paradigm. Thecultures were prepared by dissecting the midbrain floor plate fromgestation day 14 Wistar rat embryos. The tissue was dissociated withtrypsin and seeded at a density of 200,000 cells/cm² onpolyorthinine-laminin coated glass coverslips. The cells were maintainedin Dulbecco's Modified Eagle's medium and F12 medium containing hormonalsupplements (N1). The cultures were fixed with paraformaldehyde after 8days in vitro and were processed for tyrosine hydroxylase, a specificmarker for dopaminergic neurons, immunohistochemical staining. FIG. 14.FGF-13 Increases the Number of Tyrosine Hydroxylase ImmunopositiveNeurons. Dissociated cell cultures were prepared from embryonic rats.The culture medium was changed every third day and the factors were alsoadded at that time.

Parkinson's Conclusion

Since the dopaminergic neurons were isolated from animals at gestationday 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, the increase in thenumber of tyrosine hydroxylase immunopositive neurons represents anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, FGF-13 acts to prolong the survival of dopaminergic neuronswhich is needed in Parkinson's Disease.

FGF-13 Biological Activity Conclusions

FGF-13 increases the number of cells, neurite outgrowth, and the levelof neuronal specific high-affinity GABA-uptake in cortical culturesderived from embryos at gestation day 16. The results from proliferationassays using purified hippocampal astrocytes demonstrate that FGF-13increases the amount of [³H]-thymidine incorporation in astrocytecultures. Although the cortical cell cultures are maintained inserum-free medium in order to inhibit non-neuronal cell proliferation,increases in the number of astrocytes were noted following FGF-2 andFGF-13 treatments. Thus, a portion of the increase in the number ofcells observed following FGF-13 treatment is due to the proliferation ofastrocytes. However, the robust increase in the neuronal marker(GABA-uptake) suggests that a direct neuronal response is alsooccurring.

FGF-13 acted similarly to FGF-2 in human lung fibroblasts to stimulateproliferation. Neither had any effect on IL-6 and PGE₂ release fromfibroblasts but both acted synergisticly with IL-1α. In contrast toFGF-2, however, FGF-13 did not stimulate proliferation in the dermalmicrovascular endothelial cells. Dopaminergic neuron survival wasincreased by FGF-13 administration indicating it may have thereapeuticbenefits in Parkinson's Disease.

Example 6 Expression and Purification of FGF-13 in E. coli

The bacterial expression vector pHE-4 or pHE-4-5 is used for bacterialexpression in this example. pHE-4 encodes kanamycin antibioticresistance (“Kan”) and contains a bacterial origin of replication(“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”),and suitable single restriction enzyme cleavage sites. These elementsare arranged such that a DNA fragment encoding a polypeptide may beinserted in such a way as to produce that polypeptide corresponding tothe DNA fragment.

The novel pHE4 series of bacterial expression vectors, in particular,the pHE4-5 vector may be used for bacterial expression in this example.(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311). Theexpression plasmid pHE4-5/MPIFΔ23 vector plasmid DNA contains an insertwhich encodes another ORF. The construct was deposited with the AmericanType Culture Collection, 10801 University Boulevard, Manassas, Va.20110-2209, on Sep. 30, 1997 and given Accession No. 209311. Using theNde I and Asp 718 restriction sites flanking the irrelevant MPIF ORFinsert, one of ordinary skill in the art could easily use currentmolecular biological techniques to replace the irrelevant ORF in thepHE4-5 vector with the FGF-13 ORF of the present invention.

The pHE4-5 bacterial expression vector includes a neomycinphosphotransferase gene for selection, an E. coli origin of replication,a T5 phage promoter sequence, two lac operator sequences, aShine-Delgarno sequence, and the lactose operon repressor gene (lacIq).These elements are arranged such that an inserted DNA fragment encodinga polypeptide expresses that polypeptide with the six His residues(i.e., a “6×His tag”) covalently linked to the amino terminus of thatpolypeptide. The promoter and operator sequences of the pHE4-5 vectorwere made synthetically. Synthetic production of nucleic acid sequencesis well known in the art (CLONETECH 95/96 Catalog, pages 215-216,CLONETECH, 1020 East Meadow Circle, Palo Alto, Calif. 94303).

The DNA sequence encoding the desired portion of the mature FGF-13protein (i.e., amino acids 20-193 of SEQ ID NO:2) was amplified from thedeposited cDNA clone using PCR oligonucleotide primers which anneal tothe amino terminal sequences encoding the desired portion of the FGF-13protein and to sequences in the deposited construct 3′ to the cDNAcoding sequence. Additional nucleotides containing restrictions sites tofacilitate cloning in the pHE-4 vector were added to the 5′ and 3′sequences respectively.

A. For cloning the delta 42 form of FGF-13 protein, the 5′ primer hadthe sequence 5′ GGG AAT TCC ATA TGA CCG ACC AGC TGA GCA GG (SEQ IDNO:19) containing the underlined Nde I restriction site, which includesan initiation codon and following the initiation codon, 19 nucleotidesof the amino terminal coding sequence of the FGF-13 sequence in SEQ IDNO:2. One of ordinary skill in the art would appreciate, of course, thatthe point in the protein coding sequence where the 5′ primer begins maybe varied to amplify a desired portion of the complete protein shorteror longer than the mature form. The 3′ primer had the sequenceGCCCGGGGTACCTTACGTGAGGGGCTGGGGCCG (SEQ ID NO:20 containing theunderlined ASP 718 restriction site followed by a stop codon and 18nucleotides complementary to the 3′ end of the coding sequence in theFGF-13 DNA sequence in SEQ ID NO:1.

B. For cloning the 3″ delta 9 form of FGF-13 protein, the 5′ primer hadthe sequence 5′ GGGAATTCCATATGCAGGGGGAGAATCACCCGTCT 3′ (SEQ ID NO:21)containing the underlined Nde I restriction site, which includes aninitiation codon and following the initiation codon, 18 nucleotides ofthe amino terminal coding sequence of the FGF-13 sequence in SEQ IDNO:2. One of ordinary skill in the art would appreciate, of course, thatthe point in the protein coding sequence where the 5′ primer begins maybe varied to amplify a desired portion of the complete protein shorteror longer than the mature form. The 3′ primer had the sequenceGCCCGGGGTACCTTACTTGGTCCGACGGGTGGG (SEQ ID NO:22) containing theunderlined ASP 718 restriction site followed by a stop codon and 18nucleotides complementary to the 3′ end of the coding sequence in theFGF-13 DNA sequence in SEQ ID NO:1.

C. In the mature form of FGF-13, there exists a codon coding for amethione at amino acid position 20 according the SEQ ID NO:2. Upstreamof this ATG is a nucleotide sequence that may promote ribosome bindingin E. coli (Shine-Delgarno sequence). To prevent alternate ATG usage inE. coli, this sequence was mutated using a 5′ primer of the sequenceGGGAATTCCATATGCAGGGGGAGAATCACCCGTCTCCTAATTTTAACCAGTACGTGCGTGACCAGGGCGCCATG(SEQ ID NO 23) containing the underlined Nde I restriction site whichincludes an initiation codon and following the initiation codon 60nucleotides of the amino terminal coding sequence of the FGF-13 sequenceis SEQ ID NO:2. The 3′ primer has the sequence of either SEQ ID NO:20 orSEQ ID NO:22.

The amplified FGF-13 DNA fragments and the vector pHE-4 were digestedwith Nde I and ASP 718 and the digested DNAs were then ligated together.Insertion of the FGF-13 DNA into the restricted pHE-4 vector places theFGF-13 protein coding region including its associated stop codondownstream from the IPTG-inducible promoter and in-frame with theinitiating AUG in the 5′ primer.

The ligation mixture was transformed into competent E. coli cells usingstandard procedures such as those described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strain DH5αwas used in carrying out the illustrative example described herein. Thisstrain, which is only one of many that are suitable for expressingFGF-13 protein, is available commercially from Life Technologies, Inc.,Rockville, Md. Transformants were identified by their ability to grow onLB plates in the presence of kanamycin. Plasmid DNA was isolated fromresistant colonies and the identity of the cloned DNA confirmed byrestriction analysis, PCR and DNA screening.

Clones containing the desired constructs were grown overnight (“O/N”) inliquid culture in LB media supplemented with kanamycin (25 μg/ml). TheO/N culture was used to inoculate a large culture, at a dilution ofapproximately 1:25 to 1:250. The cells are grown to an optical densityat 600 nm (“OD⁶⁰⁰”) of between 0.4 and 0.6.Isopropyl-b-d-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacI repressor. Cellssubsequently were incubated further for 3 to 4 hours and were thenharvested by centrifugation.

Example 7 Terminal Deletion Variants of FGF-13

Amino and/or Carboxy terminal deletion variants of FGF-13 may beprepared using the primers disclosed in A-D to produce the variantsdescribed below. The restriction enzyme sites created are indicated.These variants may be produced using the expression systems disclosed inExample 6.

A. Deletion Variant 1 (FGF13 primers & construct seqs) 5′ delta 42/3′delta 9

5′ Nde I delta 42 ggg aat tcc ata tga ccg acc agc tga gca gg (SEQ ID NO24)

3′ delta 9 Asp 718 (6335) gcc cgg ggt acc tta ctt ggt ccg acg ggt ggg(SEQ ID NO 25)

ATGACCGACCAGCTGAGCAGGCGGCAGATCCGCGAGTACCAACTCTACAGCAGGACCAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGCAACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCAAAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAAGCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTATACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGCGGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCGCCTCTACCAAGGCCAGCTGCCCTTCCCCAACCACGCCGAGAAGCAGAAGCAGTTCGAGTTTGTGGGCTCCGCCCCCACCCGTCGGACCAAGTAA(SEQ ID NO 26)

MTDQLSRRQIREYQLYSRTSGKHVQVTGRRISATAEDGNKFAKLIVETDTFGSRVRIKGAESEKYICMNKRGKLIGKPSGKSKDCVFTEIVLENNYTAFQNARHEGWFMAFTRQGRPRQASRSRQNQREAHFIKRLYQGQLPFPNHAEKQKQFEFVGSAPTRRTK.(SEQ ID NO 27)

B. Deletion Variant 2 (5′ delta 42/3′ full)

5′ Nde I delta 42 ggg aat tcc ata tga ccg acc agc tga gca gg (SEQ ID NO28)

3′ full Asp 718 (6638) gcc cgg ggt acc tta cgt gag ggg ctg ggg ccg (SEQID NO 29)

ATGACCGACCAGCTGAGCAGGCGGCAGATCCGCGAGTACCAACTYTACAGCAGGACCAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGCAACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCAAAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAAGCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTATACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGCGGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCGCCTCTACCAAGGCCAGCTGCCCTTCCCCAACCACGCCGAGAAGCAGAAGCAGTTCGAGTTTGTGGGCTCCGCCCCCACCCGYCGGACCAAGCGCACACGGCGGCCCCAGCCCCTCACGTAA(SEQ ID NO 30)

MTDQLSRRQIREYQLYSRTSGKHVQVTGRRISATAEDGNKFAKLIVETDTFGSRVRIKGAESEKYICMNKRGKLIGKPSGKSKDCVFTEIVLENNYTAFQNARHEGWFMAFTRQGRPRQASRSRQNQREAHFIKRLYQGQLPFPNHAEKQKQFEFVGSAPTRRTKRTRRPQPLT.(SEQ ID NO 31)

C. Deletion Variant 3 (5′ delta23/3′ full)

5′ NdeI Delta 23 SDG (6881) deletion ggg aat tcc ata tgc agg ggg aga atcacc cgt ctc cta att tta acc agt acg tgc gtg acc agg gcg cca tg (SEQ IDNO 32)

3′ delta 9 Asp 718 (6335) gcc cgg ggt acc tta ctt ggt ccg acg ggt ggg(SEQ ID NO 33)

ATGCAGGGGGAGAATCACCCGTCTCCTAATTTTAACCAGTACGTGCGTGACCAGGGCGCCATGACCGACCAGCTGAGCAGGCGGCAGATCCGCGAGTACCAACTCTACAGCAGGACCAGTGGCAAGCACGTGCAGGTCACCGGGCGTCGCATCTCCGCCACCGCCGAGGACGGCAACAAGTTTGCCAAGCTCATAGTGGAGACGGACACGTTTGGCAGCCGGGTTCGCATCAAAGGGGCTGAGAGTGAGAAGTACATCTGTATGAACAAGAGGGGCAAGCTCATCGGGAAGCCCAGCGGGAAGAGCAAAGACTGCGTGTTCACGGAGATCGTGCTGGAGAACAACTATACGGCCTTCCAGAACGCCCGGCACGAGGGCTGGTTCATGGCCTTCACGCGGCAGGGGCGGCCCCGCCAGGCTTCCCGCAGCCGCCAGAACCAGCGCGAGGCCCACTTCATCAAGCGCCTCTACCAAGGCCAGCTGCCCTTCCCCAACCACGCCGAGAAGCAGAAGCAGTTCGAGTTTGTGGGCTCCGCCCCCACCCGTCGGACCAAGTAA(SEQ ID NO 34)

MQGENHPSPNFNQYVRDQGAMTDQLSRRQIREYQLYSRTSGKHVQVTGRRISATAEDGNKFAKLIVETDTFGSRVRIKGAESEKYICMNKRGKLIGKPSGKSKDCVFTEIVLENNYTAFQNARHEGWFMAFTRQGRPRQASRSRQNQREAHFIKRLYQGQLPFPNHAEKQKQFEFVGSAPTRRTK.(SEQ ID NO 35)

D. Deletion Variant 4 (5′ delta 23 SDG deletion/3′ full)

5′ NdeI Delta 23 SDG (6881) deletion GGG AAT TCC ATA TGC AGG GGG AGA ATCACC CGT CTC CTA ATT TTA ACC AGT ACG TGC GTG ACC AGG GCG CCA TG (SEQ IDNO 36) 3′ FULL ASP 718 (6638) GCC CGG GGT ACC TTA CGT GAG GGG CTG GGGCCG (SEQ ID NO 37)

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims. The entire disclosure of all publications(including patents, patent applications, journal articles, laboratorymanuals, books, or other documents) cited herein are hereby incorporatedby reference.

37 1209 base pairs nucleic acid single linear DNA (genomic) CDS 1..648mat_peptide 70..648 sig_peptide 1..67 1 ATG GGA GCC GCC CGC CTG CTG CCCAAC CTC ACT CTG TGC TTA CAG CTG 48 Met Gly Ala Ala Arg Leu Leu Pro AsnLeu Thr Leu Cys Leu Gln Leu -23 -20 -15 -10 CTG ATT CTC TGC TGT CAA ACTCAG GGG GAG AAT CAC CCG TCT CCT AAT 96 Leu Ile Leu Cys Cys Gln Thr GlnGly Glu Asn His Pro Ser Pro Asn -5 -1 1 5 TTT AAC CAG TAC GTG AGG GACCAG GGC GCC ATG ACC GAC CAG CTG AGC 144 Phe Asn Gln Tyr Val Arg Asp GlnGly Ala Met Thr Asp Gln Leu Ser 10 15 20 25 AGG CGG CAG ATC CGC GAG TACCAA CTC TAC AGC AGG ACC AGT GGC AAG 192 Arg Arg Gln Ile Arg Glu Tyr GlnLeu Tyr Ser Arg Thr Ser Gly Lys 30 35 40 CAC GTG CAG GTC ACC GGG CGT CGCATC TCC GCC ACC GCC GAG GAC GGC 240 His Val Gln Val Thr Gly Arg Arg IleSer Ala Thr Ala Glu Asp Gly 45 50 55 AAC AAG TTT GCC AAG CTC ATA GTG GAGACG GAC ACG TTT GGC AGC CGG 288 Asn Lys Phe Ala Lys Leu Ile Val Glu ThrAsp Thr Phe Gly Ser Arg 60 65 70 GTT CGC ATC AAA GGG GCT GAG AGT GAG AAGTAC ATC TGT ATG AAC AAG 336 Val Arg Ile Lys Gly Ala Glu Ser Glu Lys TyrIle Cys Met Asn Lys 75 80 85 AGG GGC AAG CTC ATC GGG AAG CCC AGC GGG AAGAGC AAA GAC TGC GTG 384 Arg Gly Lys Leu Ile Gly Lys Pro Ser Gly Lys SerLys Asp Cys Val 90 95 100 105 TTC ACG GAG ATC GTG CTG GAG AAC AAC TATACG GCC TTC CAG AAC GCC 432 Phe Thr Glu Ile Val Leu Glu Asn Asn Tyr ThrAla Phe Gln Asn Ala 110 115 120 CGG CAC GAG GGC TGG TTC ATG GCC TTC ACGCGG CAG GGG CGG CCC CGC 480 Arg His Glu Gly Trp Phe Met Ala Phe Thr ArgGln Gly Arg Pro Arg 125 130 135 CAG GCT TCC CGC AGC CGC CAG AAC CAG CGCGAG GCC CAC TTC ATC AAG 528 Gln Ala Ser Arg Ser Arg Gln Asn Gln Arg GluAla His Phe Ile Lys 140 145 150 CGC CTC TAC CAA GGC CAG CTG CCC TTC CCCAAC CAC GCC GAG AAG CAG 576 Arg Leu Tyr Gln Gly Gln Leu Pro Phe Pro AsnHis Ala Glu Lys Gln 155 160 165 AAG CAG TTC GAG TTT GTG GGC TCC GCC CCCACC CGC CGG ACC AAG CGC 624 Lys Gln Phe Glu Phe Val Gly Ser Ala Pro ThrArg Arg Thr Lys Arg 170 175 180 185 ACA CGG CGG CCC CAG CCC CTC ACGTAGTCTGGGA GGCAGGGGGC AGCAGCCCCT 678 Thr Arg Arg Pro Gln Pro Leu Thr 190GGGCCGCCTC CCCACCCCTT TCCCTTCTTA ATCCAAGGAC TGGGCTGGGG TGGCGGGAGG 738GGAGCCAGAT CCCCGAGGGA GGACCCTGAG GGCCGCGAAG CATCCGAGCC CCCAGCTGGG 798AAGGGGCAGG CCGGTGCCCC AGGGGCGGCT GGCACAGTGC CCCCTTCCCG GACGGGTGGC 858AGGCCCTGGA GAGGAACTGA GTGTCACCCT GATCTCAGGC CACCAGCCTC TGCCGGCCTC 918CCAGCCGGGC TCCTGAAGCC CGCTGAAAGG TCAGCGACTG AAGGCCTTGC AGACAACCGT 978CTGGAGGTGG CTGTCCTCAA AATCTGCTTC TCGGATCTCC CTCAGTCTGC CCCCAGCCCC 1038CAAACTCCTC CTGGCTAGAC TGTAGGAAGG GACTTTTGTT TGTTTGTTTG TTTCAGGAAA 1098AAAGAAAGGG AGAGAGAGGA AAATAGAGGG TTGTCCACTC CTCACATTCC ACGACCCAGG 1158CCTGCACCCC ACCCCCAACT CCCAGCCCCG GAATAAAACC ATTTTCCTGC A 1209 216 aminoacids amino acid linear protein 2 Met Gly Ala Ala Arg Leu Leu Pro AsnLeu Thr Leu Cys Leu Gln Leu -23 -20 -15 -10 Leu Ile Leu Cys Cys Gln ThrGln Gly Glu Asn His Pro Ser Pro Asn -5 -1 1 5 Phe Asn Gln Tyr Val ArgAsp Gln Gly Ala Met Thr Asp Gln Leu Ser 10 15 20 25 Arg Arg Gln Ile ArgGlu Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys 30 35 40 His Val Gln Val ThrGly Arg Arg Ile Ser Ala Thr Ala Glu Asp Gly 45 50 55 Asn Lys Phe Ala LysLeu Ile Val Glu Thr Asp Thr Phe Gly Ser Arg 60 65 70 Val Arg Ile Lys GlyAla Glu Ser Glu Lys Tyr Ile Cys Met Asn Lys 75 80 85 Arg Gly Lys Leu IleGly Lys Pro Ser Gly Lys Ser Lys Asp Cys Val 90 95 100 105 Phe Thr GluIle Val Leu Glu Asn Asn Tyr Thr Ala Phe Gln Asn Ala 110 115 120 Arg HisGlu Gly Trp Phe Met Ala Phe Thr Arg Gln Gly Arg Pro Arg 125 130 135 GlnAla Ser Arg Ser Arg Gln Asn Gln Arg Glu Ala His Phe Ile Lys 140 145 150Arg Leu Tyr Gln Gly Gln Leu Pro Phe Pro Asn His Ala Glu Lys Gln 155 160165 Lys Gln Phe Glu Phe Val Gly Ser Ala Pro Thr Arg Arg Thr Lys Arg 170175 180 185 Thr Arg Arg Pro Gln Pro Leu Thr 190 155 amino acids aminoacid single linear protein 3 Met Ala Glu Gly Glu Ile Thr Thr Phe Thr AlaLeu Thr Glu Lys Phe 1 5 10 15 Asn Leu Pro Pro Gly Asn Tyr Lys Lys ProLys Leu Leu Tyr Cys Ser 20 25 30 Asn Gly Gly His Phe Leu Arg Ile Leu ProAsp Gly Thr Val Asp Gly 35 40 45 Thr Arg Asp Arg Ser Asp Gln His Ile GlnLeu Gln Leu Ser Ala Glu 50 55 60 Ser Val Gly Glu Val Tyr Ile Lys Ser ThrGlu Thr Gly Gln Tyr Leu 65 70 75 80 Ala Met Asp Thr Asp Gly Leu Leu TyrGly Ser Gln Thr Pro Asn Glu 85 90 95 Glu Cys Leu Phe Leu Glu Arg Leu GluGlu Asn His Tyr Asn Thr Tyr 100 105 110 Ile Ser Lys Lys His Ala Glu LysAsn Trp Phe Val Gly Leu Lys Lys 115 120 125 Asn Gly Ser Cys Lys Arg GlyPro Arg Thr His Tyr Gly Gln Lys Ala 130 135 140 Ile Leu Phe Leu Pro LeuPro Val Ser Ser Asp 145 150 155 155 amino acids amino acid single linearprotein 4 Met Ala Ala Gly Ser Ile Thr Thr Leu Pro Ala Leu Pro Glu AspGly 1 5 10 15 Gly Ser Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro LysArg Leu 20 25 30 Tyr Cys Lys Asn Gly Gly Phe Phe Leu Arg Ile His Pro AspGly Arg 35 40 45 Val Asp Gly Val Arg Glu Lys Ser Asp Pro His Ile Lys LeuGln Leu 50 55 60 Gln Ala Glu Glu Arg Gly Val Val Ser Ile Lys Gly Val CysAla Asn 65 70 75 80 Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu AlaSer Lys Cys 85 90 95 Val Thr Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu SerAsn Asn Tyr 100 105 110 Asn Thr Tyr Arg Ser Arg Lys Tyr Thr Ser Trp TyrVal Ala Leu Lys 115 120 125 Arg Thr Gly Gln Tyr Lys Leu Gly Ser Lys ThrGly Pro Gly Gln Lys 130 135 140 Ala Ile Leu Phe Leu Pro Met Ser Ala LysSer 145 150 155 239 amino acids amino acid single linear protein 5 MetGly Leu Ile Trp Leu Leu Leu Leu Ser Leu Leu Glu Pro Gly Trp 1 5 10 15Pro Ala Ala Gly Pro Gly Ala Arg Leu Arg Arg Asp Ala Gly Gly Arg 20 25 30Gly Gly Val Tyr Glu His Leu Gly Gly Ala Pro Arg Arg Arg Lys Leu 35 40 45Tyr Cys Ala Thr Lys Tyr His Leu Gln Leu His Pro Ser Gly Arg Val 50 55 60Asn Gly Ser Leu Glu Asn Ser Ala Tyr Ser Ile Leu Glu Ile Thr Ala 65 70 7580 Val Glu Val Gly Ile Val Ala Ile Arg Gly Leu Phe Ser Gly Arg Tyr 85 9095 Leu Ala Met Asn Lys Arg Gly Arg Leu Tyr Ala Ser Glu His Tyr Ser 100105 110 Ala Glu Cys Glu Phe Val Glu Arg Ile His Glu Leu Gly Tyr Asn Thr115 120 125 Tyr Ala Ser Arg Leu Tyr Arg Thr Val Ser Ser Thr Pro Gly AlaArg 130 135 140 Arg Gln Pro Ser Ala Glu Arg Leu Trp Tyr Val Ser Val AsnGly Lys 145 150 155 160 Gly Arg Pro Arg Arg Gly Phe Lys Thr Arg Arg ThrGln Lys Ser Ser 165 170 175 Leu Phe Leu Pro Arg Val Leu Asp His Arg AspHis Glu Met Val Arg 180 185 190 Gln Leu Gln Ser Gly Leu Pro Arg Pro ProGly Lys Gly Val Gln Pro 195 200 205 Arg Arg Arg Arg Gln Lys Gln Ser ProAsp Asn Leu Glu Pro Ser His 210 215 220 Val Gln Ala Ser Arg Leu Gly SerGln Leu Glu Ala Ser Ala His 225 230 235 206 amino acids amino acidsingle linear protein 6 Met Ser Gly Pro Gly Thr Ala Ala Val Ala Leu LeuPro Ala Val Leu 1 5 10 15 Leu Ala Leu Leu Ala Pro Trp Ala Gly Arg GlyGly Ala Ala Ala Pro 20 25 30 Thr Ala Pro Asn Gly Thr Leu Glu Ala Glu LeuGlu Arg Arg Trp Glu 35 40 45 Ser Leu Val Ala Leu Ser Leu Ala Arg Leu ProVal Ala Ala Gln Pro 50 55 60 Lys Glu Ala Ala Val Gln Ser Gly Ala Gly AspTyr Leu Leu Gly Ile 65 70 75 80 Lys Arg Leu Arg Arg Leu Tyr Cys Asn ValGly Ile Gly Phe His Leu 85 90 95 Gln Ala Leu Pro Asp Gly Arg Ile Gly GlyAla His Ala Asp Thr Arg 100 105 110 Asp Ser Leu Leu Glu Leu Ser Pro ValGlu Arg Gly Val Val Ser Ile 115 120 125 Phe Gly Val Ala Ser Arg Phe PheVal Ala Met Ser Ser Lys Gly Lys 130 135 140 Leu Tyr Gly Ser Pro Phe PheThr Asp Glu Cys Thr Phe Lys Glu Ile 145 150 155 160 Leu Leu Pro Asn AsnTyr Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly 165 170 175 Met Phe Ile AlaLeu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn Arg 180 185 190 Val Ser ProThr Met Lys Val Thr His Phe Leu Pro Arg Leu 195 200 205 267 amino acidsamino acid single linear protein 7 Met Ser Leu Ser Phe Leu Leu Leu LeuPhe Phe Ser His Leu Ile Leu 1 5 10 15 Ser Ala Trp Ala His Gly Glu LysArg Leu Ala Pro Lys Gly Gln Pro 20 25 30 Gly Pro Ala Ala Thr Asp Arg AsnPro Arg Gly Ser Ser Ser Arg Gln 35 40 45 Ser Ser Ser Ser Ala Met Ser SerSer Ser Ala Ser Ser Ser Pro Ala 50 55 60 Ala Ser Leu Gly Ser Gln Gly SerGly Leu Glu Gln Ser Ser Phe Gln 65 70 75 80 Trp Ser Leu Gly Ala Arg ThrGly Ser Leu Tyr Cys Arg Val Gly Ile 85 90 95 Gly Phe His Leu Gln Ile TyrPro Asp Gly Lys Val Asn Gly Ser His 100 105 110 Glu Ala Asn Met Leu SerVal Leu Glu Ile Phe Ala Val Ser Gln Gly 115 120 125 Ile Val Gly Ile ArgGly Val Phe Ser Asn Lys Phe Leu Ala Met Ser 130 135 140 Lys Lys Gly LysLeu His Ala Ser Ala Lys Phe Thr Asp Asp Cys Lys 145 150 155 160 Phe ArgGlu Arg Phe Gln Glu Asn Ser Tyr Asn Thr Tyr Ala Ser Ala 165 170 175 IleHis Arg Thr Glu Lys Thr Gly Arg Glu Trp Tyr Val Ala Leu Asn 180 185 190Lys Arg Gly Lys Ala Lys Arg Gly Cys Ser Pro Arg Val Lys Pro Gln 195 200205 His Ile Ser Thr His Phe Leu Pro Arg Phe Lys Gln Ser Glu Gln Pro 210215 220 Glu Leu Ser Phe Thr Val Thr Val Pro Glu Lys Lys Asn Pro Pro Ser225 230 235 240 Pro Ile Lys Ser Lys Ile Pro Leu Ser Ala Pro Arg Lys AsnThr Asn 245 250 255 Ser Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 260 265208 amino acids amino acid single linear protein 8 Met Ala Leu Gly GlnLys Leu Phe Ile Thr Met Ser Arg Gly Ala Gly 1 5 10 15 Arg Leu Gln GlyThr Leu Trp Ala Leu Val Phe Leu Gly Ile Leu Val 20 25 30 Gly Met Val ValPro Ser Pro Ala Gly Thr Arg Ala Asn Asn Thr Leu 35 40 45 Leu Asp Ser ArgGly Trp Gly Thr Leu Leu Ser Arg Ser Arg Ala Gly 50 55 60 Leu Ala Gly GluIle Ala Gly Val Asn Trp Glu Ser Gly Tyr Leu Val 65 70 75 80 Gly Ile LysArg Gln Arg Arg Leu Tyr Cys Asn Val Gly Ile Gly Phe 85 90 95 His Leu GlnVal Leu Pro Asp Gly Arg Ile Ser Gly Thr His Glu Glu 100 105 110 Asn ProTyr Ser Leu Leu Glu Ile Ser Thr Val Glu Arg Gly Val Val 115 120 125 SerLeu Phe Gly Val Arg Ser Ala Leu Phe Val Ala Met Asn Ser Lys 130 135 140Gly Arg Leu Tyr Ala Thr Pro Ser Phe Gln Glu Glu Cys Lys Phe Arg 145 150155 160 Glu Thr Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu Ser Asp Leu Tyr165 170 175 Gln Gly Thr Tyr Ile Ala Leu Ser Lys Tyr Gly Arg Val Lys ArgGly 180 185 190 Ser Lys Val Ser Pro Ile Met Thr Val Thr His Phe Leu ProArg Ile 195 200 205 194 amino acids amino acid single linear protein 9Met His Lys Trp Ile Leu Thr Trp Ile Leu Pro Thr Leu Leu Tyr Arg 1 5 1015 Ser Cys Phe His Ile Ile Cys Leu Val Gly Thr Ile Ser Leu Ala Cys 20 2530 Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Asn Cys Ser Ser 35 4045 Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp Ile 50 5560 Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg Ile Asp 65 7075 80 Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn 8590 95 Ile Met Glu Ile Arg Thr Val Ala Val Gly Ile Val Ala Ile Lys Gly100 105 110 Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys LeuTyr 115 120 125 Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu LeuIle Leu 130 135 140 Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp ThrHis Asn Gly 145 150 155 160 Gly Glu Met Phe Val Ala Leu Asn Gln Lys GlyIle Pro Val Arg Gly 165 170 175 Lys Lys Thr Lys Lys Glu Gln Lys Thr AlaHis Phe Leu Pro Met Ala 180 185 190 Ile Thr 215 amino acids amino acidsingle linear protein 10 Met Gly Ser Pro Arg Ser Ala Leu Ser Cys Leu LeuLeu His Leu Leu 1 5 10 15 Val Leu Cys Leu Gln Ala Gln Val Thr Val GlnSer Ser Pro Asn Phe 20 25 30 Thr Gln His Val Arg Glu Gln Ser Leu Val ThrAsp Gln Leu Ser Arg 35 40 45 Arg Leu Ile Arg Thr Tyr Gln Leu Tyr Ser ArgThr Ser Gly Lys His 50 55 60 Val Gln Val Leu Ala Asn Lys Arg Ile Asn AlaMet Ala Glu Asp Gly 65 70 75 80 Asp Pro Phe Ala Lys Leu Ile Val Glu ThrAsp Thr Phe Gly Ser Arg 85 90 95 Val Arg Val Arg Gly Ala Glu Thr Gly LeuTyr Ile Cys Met Asn Lys 100 105 110 Lys Gly Lys Leu Ile Ala Lys Ser AsnGly Lys Gly Lys Asp Cys Val 115 120 125 Phe Thr Glu Ile Val Leu Glu AsnAsn Tyr Thr Ala Leu Gln Asn Ala 130 135 140 Lys Tyr Glu Gly Trp Tyr MetAla Phe Thr Arg Lys Gly Arg Pro Arg 145 150 155 160 Lys Gly Ser Lys ThrArg Gln His Gln Arg Glu Val His Phe Met Lys 165 170 175 Arg Leu Pro ArgGly His His Thr Thr Glu Gln Ser Leu Arg Phe Glu 180 185 190 Phe Leu AsnTyr Pro Pro Phe Thr Arg Ser Leu Arg Gly Ser Gln Arg 195 200 205 Thr TrpAla Pro Glu Pro Arg 210 215 32 base pairs nucleic acid single linear DNA(genomic) 11 GCCAGACCAT GGAGAATCAC CCGTCTCCTA AT 32 30 base pairsnucleic acid single linear DNA (genomic) 12 GATTTAAGAT CTCGTGAGGGGCTGGGGCCG 30 32 base pairs nucleic acid single linear DNA (genomic) 13CTAGTCGCAT GCAGGGGGAG AATCACCCGT CT 32 33 base pairs nucleic acid singlelinear DNA (genomic) 14 GCTTGAAAGC TTCTACGTGA GGGGCTGGGG CCG 33 30 basepairs nucleic acid single linear DNA (genomic) 15 CTAGTGGATC CCGAGAATCACCCGTCTCCT 30 30 base pairs nucleic acid single linear DNA (genomic) 16CGACTTCTAG AACCTCGGGG ATCTGGCTCC 30 136 base pairs nucleic acid singlelinear DNA (genomic) 17 CTAGCCGGAT CCGCCACCAT GAACTCCTTC TCCACAAGCGCCTTCGGTCC AGTTGCCTTC 60 TCCCTGGGGC TGCTCCTGGT GTTGCCTGCT GCCTTCCCTGCCCCAGTTGT GAGACCAGGG 120 GGAGAATCAC CCGTCT 136 30 base pairs nucleicacid single linear DNA (genomic) 18 GCTTGATCTA GACGTGAGGG GCTGGGGCCG 3032 base pairs nucleic acid single linear DNA (genomic) 19 GGGAATTCCATATGACCGACC AGCTGAGCAG G 32 33 base pairs nucleic acid single linear DNA(genomic) 20 GCCCGGGGTA CCTTACGTGA GGGGCTGGGG CCG 33 35 base pairsnucleic acid single linear DNA (genomic) 21 GGGAATTCCA TATGCAGGGGGAGAATCACC CGTCT 35 33 base pairs nucleic acid single linear DNA(genomic) 22 GCCCGGGGTA CCTTACTTGG TCCGACGGGT GGG 33 74 base pairsnucleic acid single linear DNA (genomic) 23 GGGAATTCCA TATGCAGGGGGAGAATCACC CGTCTCCTAA TTTTAACCAG TACGTGCGTG 60 ACCAGGGCGC CATG 74 32base pairs nucleic acid single linear DNA (genomic) 24 GGGAATTCCATATGACCGAC CAGCTGAGCA GG 32 33 base pairs nucleic acid single linear DNA(genomic) 25 GCCCGGGGTA CCTTACTTGG TCCGACGGGT GGG 33 497 base pairsnucleic acid single linear DNA (genomic) 26 ATGACCGACC AGCTGAGCAGGCGGCAGATC CGCGAGTACC AACTCTACAG CAGGACCAGT 60 GGCAAGCACG TGCAGGTCACCGGGCGTCGC ATCTCCGCCA CCGCCGAGGA CGGCAACAAG 120 TTTGCCAAGC TCATAGTGGAGACGGACACG TTTGGCAGCC GGGTTCGCAT CAAAGGGGCT 180 GAGAGTGAGA AGTACATCTGTATGAACAAG AGGGGCAAGC TCATCGGGAA GCCCAGCGGG 240 AAGAGCAAAG ACTGCGTGTTCACGGAGATC GTGCTGGAGA ACAACTATAC GGCCTTCCAG 300 AACGCCCGGC ACGAGGGCTGGTTCATGGCC TTCACGCGGC AGGGGCGGCC CCGCCAGGCT 360 TCCCGCAGCC GCCAGAACCAGCGCGAGGCC CACTTCATCA AGCGCTCTAC CAAGGCCAGC 420 TGCCCTTCCC CAACCACGCCGAGAAGCAGA AGCAGTTCGA GTTTGTGGGC TCCGCCCCCA 480 CCCGTCGGAC CAAGTAA 497164 amino acids amino acid single linear protein 27 Met Thr Asp Gln LeuSer Arg Arg Gln Ile Arg Glu Tyr Gln Leu Tyr 1 5 10 15 Ser Arg Thr SerGly Lys His Val Gln Val Thr Gly Arg Arg Ile Ser 20 25 30 Ala Thr Ala GluAsp Gly Asn Lys Phe Ala Lys Leu Ile Val Glu Thr 35 40 45 Asp Phe Gly SerArg Val Arg Ile Lys Gly Ala Glu Ser Glu Lys Tyr 50 55 60 Ile Cys Met AsnLys Arg Gly Lys Leu Ile Gly Lys Pro Ser Gly Lys 65 70 75 80 Ser Lys AspCys Val Phe Thr Glu Ile Val Leu Glu Asn Asn Tyr Thr 85 90 95 Ala Phe GlnAsn Ala Arg His Glu Gly Trp Phe Met Ala Phe Thr Arg 100 105 110 Gln GlyArg Pro Arg Gln Ala Ser Arg Ser Arg Gln Asn Gln Arg Glu 115 120 125 AlaHis Phe Ile Lys Arg Leu Tyr Gln Gly Gln Leu Pro Phe Pro Asn 130 135 140His Ala Glu Lys Gln Lys Gln Phe Glu Phe Val Gly Ser Ala Pro Thr 145 150155 160 Arg Arg Thr Lys 32 base pairs nucleic acid single linear DNA(genomic) 28 GGGAATTCCA TATGACCGAC CAGCTGAGCA GG 32 33 base pairsnucleic acid single linear DNA (genomic) 29 GCCCGGGGTA CCTTACGTGAGGGGCTGGGG CCG 33 524 base pairs nucleic acid single linear DNA(genomic) 30 ATGACCGACC AGCTGAGCAG GCGGCAGATC CGCGAGTACC AACTTACAGCAGGACCAGTG 60 GCAAGCACGT GCAGGTCACC GGGCGTCGCA TCTCCGCCAC CGCCGAGGACGGCAACAAGT 120 TTGCCAAGCT CATAGTGGAG ACGGACACGT TTGGCAGCCG GGTTCGCATCAAAGGGGCTG 180 AGAGTGAGAA GTACATCTGT ATGAACAAGA GGGGCAAGCT CATCGGGAAGCCCAGCGGGA 240 AGAGCAAAGA CTGCGTGTTC ACGGAGATCG TGCTGGAGAA CAACTATACGGCCTTCCAGA 300 ACGCCCGGCA CGAGGGCTGG TTCATGGCCT TCACGCGGCA GGGGCGGCCCCCGCCAGGCT 360 TCCCGCAGCC GCCAGAACCA GCGCGAGGCC CACTTCATCA AGCGCCTCTACCAAGGCCAG 420 CTGCCCTTCC CCAACCACGC CGAGAAGCAG AAGCAGTTCG AGTTTGTGGGCTCCGCCCCC 480 ACCCGCGGAC CAAGCGCACA CGGCGGCCCC AGCCCCTCAC GTAA 524 175amino acids amino acid single linear protein 31 Met Thr Asp Gln Leu SerArg Arg Gln Ile Arg Glu Tyr Gln Leu Tyr 1 5 10 15 Ser Arg Thr Ser GlyLys His Val Gln Val Thr Gly Arg Arg Ile Ser 20 25 30 Ala Thr Ala Glu AspGly Asn Lys Phe Ala Lys Leu Ile Val Glu Thr 35 40 45 Asp Thr Phe Gly SerArg Val Arg Ile Lys Gly Ala Glu Ser Glu Lys 50 55 60 Tyr Ile Cys Met AsnLys Arg Gly Lys Leu Ile Ile Gly Lys Pro Ser 65 70 75 80 Gly Lys Ser LysAsp Cys Val Phe Thr Glu Ile Val Leu Glu Asn Asn 85 90 95 Tyr Thr Ala PheGln Asn Ala Arg His Glu Gly Trp Phe Met Ala Phe 100 105 110 Thr Arg GlnGly Arg Pro Arg Gln Ala Ser Arg Ser Arg Gln Asn Gln 115 120 125 Arg GluAla His Phe Ile Lys Arg Leu Tyr Gln Gly Gln Leu Pro Phe 130 135 140 ProAsn His Ala Glu Lys Gln Lys Gln Phe Glu Phe Val Gly Ser Ala 145 150 155160 Pro Thr Arg Arg Thr Lys Arg Thr Arg Arg Pro Gln Pro Leu Thr 165 170175 74 base pairs nucleic acid single linear DNA (genomic) 32 GGGAATTCCATATGCAGGGG GAGAATCACC CGTCTCCTAA TTTTAACCAG TACGTGCGTG 60 ACCAGGGCGCCATG 74 33 base pairs nucleic acid single linear DNA (genomic) 33GCCCGGGGTA CCTTACTTGG TCCGACGGGT GGG 33 554 base pairs nucleic acidsingle linear DNA (genomic) 34 ATGCAGGGGG AGAATCACCC GTCTCCTAATTTTAACCAGT ACGTGCGTGA CCAGGGCGCC 60 ATGACCGACC AGCTGAGCAG GCGGCAGATCCGCGAGTACC AACTCTACAG CAGGACCAGT 120 GGCAAGCACG TGCAGGTCAC CGGGCGTCGCATCTCCGCCA CCGCCGAGGA CGGCAACAAG 180 TTTGCCAAGC TCATAGTGGA GACGGACACGTTTGGCAGCC GGGTTCGCAT CAAAGGGCTG 240 AGAGTGAGAA GTACATCTGT ATGAACAAGAGGGGCAAGCT CATCGGGAAG CCCAGCGGGA 300 AGAGCAAAGA CTGCGTGTTC ACGGAGATCGTGCTGGAGAA CAACTATACG GCTTCCAGAA 360 CGCCCGGCAC GAGGGCTGGT TCATGGCCTTCACGGGCAGG GGCGGCCCCG CCAGGCTTCC 420 CGCAGCCGCC AGAACCAGCG CGAGGCCCACTTCATCAAGC GCCTCTACCA AGGCCAGCTG 480 CCCTTCCCCA ACCACGCCGA GAAGCAGAAGCAGTTCGAGT TTGTGGCTCC GCCCCCACCC 540 GTCGGACCAA GTAA 554 185 amino acidsamino acid single linear protein 35 Met Gln Gly Glu Asn His Pro Ser ProAsn Phe Asn Gln Tyr Val Arg 1 5 10 15 Asp Gln Gly Ala Met Thr Asp GlnLeu Ser Arg Arg Gln Ile Arg Glu 20 25 30 Tyr Gln Leu Tyr Ser Arg Thr SerGly Lys His Val Gln Val Thr Gly 35 40 45 Arg Arg Ile Ser Ala Thr Ala GluAsp Gly Asn Lys Phe Ala Lys Leu 50 55 60 Ile Val Glu Thr Asp Thr Phe GlySer Arg Val Arg Ile Lys Gly Ala 65 70 75 80 Glu Ser Glu Lys Tyr Ile CysMet Asn Lys Arg Gly Lys Leu Ile Gly 85 90 95 Lys Pro Ser Gly Lys Ser LysAsp Cys Val Phe Thr Glu Ile Val Leu 100 105 110 Glu Asn Asn Tyr Thr AlaPhe Gln Asn Ala Arg His Glu Gly Trp Phe 115 120 125 Met Ala Phe Thr ArgGln Gly Arg Pro Arg Gln Ala Ser Arg Ser Arg 130 135 140 Gln Asn Gln ArgGlu Ala His Phe Ile Lys Arg Leu Tyr Gln Gly Gln 145 150 155 160 Leu ProPhe Pro Asn His Ala Glu Lys Gln Lys Gln Phe Glu Phe Val 165 170 175 GlySer Ala Pro Thr Arg Arg Thr Lys 180 185 74 base pairs nucleic acidsingle linear DNA (genomic) 36 GGGAATTCCA TATGCAGGGG GAGAATCACCCGTCTCCTAA TTTTAACCAG TACGTGCGTG 60 ACCAGGGCGC AATG 74 33 base pairsnucleic acid single linear DNA (genomic) 37 GCCCGGGGTA CCTTACGTGAGGGGCTGGGG CCG 33

What is claimed is:
 1. An isolated protein molecule comprising an aminoacid sequence selected from the group consisting of: (a) amino acids +1to +193 of SEQ ID NO:2; (b) a mature portion of the protein encoded bythe cDNA contained in ATCC Deposit No. 97148; (c) amino acids −23 to+193 of SEQ ID NO:2; and (d) the full length amino acid sequence encodedby the cDNA contained in ATCC Deposit No.
 97148. 2. The isolated proteinmolecule of claim 1, wherein said amino acid sequence is (a).
 3. Anisolated protein molecule produced by the method comprising: (a)expressing the protein molecule of claim 2 from a host cell; and (b)recovering said protein molecule.
 4. The isolated protein molecule ofclaim 1, wherein said amino acid sequence is (b).
 5. An isolated proteinmolecule produced by the method comprising: (a) expressing the proteinmolecule of claim 4 from a host cell; and (b) recovering said proteinmolecule.
 6. The isolated protein molecule of claim 1, wherein saidamino acid sequence is (c).
 7. An isolated protein molecule produced bythe method comprising: (a) expressing the protein molecule of claim 6from a host cell; and (b) recovering said protein molecule.
 8. Theisolated protein molecule of claim 1, wherein said amino acid sequenceis (d).
 9. An isolated protein molecule produced by the methodcomprising: (a) expressing the protein molecule of claim 8 from a hostcell; and (b) recovering said protein molecule.
 10. The isolated proteinmolecule of claim 1 further comprising a heterologous polypeptide.
 11. Acomposition comprising the protein molecule of claim 1 and apharmaceutically acceptable carrier.
 12. An isolated protein moleculecomprising an amino acid sequence selected from the group consisting of:(a) at least 30 contiguous amino acids as set forth in SEQ ID NO:2 orencoded by the human cDNA contained in ATCC Deposit No. 97148; (b) apolypeptide fragment of the amino acid sequence of SEQ ID NO:2 or apolypeptide fragment encoded by the human cDNA contained in ATCC DepositNo. 97148, wherein said fragment has neural cell activity; (c) aminoacids +91 to +108 of SEQ ID NO:2; and (d) amino acids −22 to +108 of SEQID NO:2.
 13. The isolated protein molecule of claim 12, wherein saidamino acid sequence is (a).
 14. The isolated protein molecule of claim13, wherein said amino acid sequence is at least 50 contiguous aminoacids as set forth in SEQ ID NO:2 or encoded by the human cDNA containedin ATCC Deposit No.
 97148. 15. An isolated protein molecule produced bythe method comprising: (a) expressing the protein molecule of claim 13from a host cell; and (b) recovering said protein molecule.
 16. Theisolated protein molecule of claim 12, wherein said amino acid sequenceis (b).
 17. An isolated protein molecule produced by the methodcomprising: (a) expressing the protein molecule of claim 16 from a hostcell; and (b) recovering said protein molecule.
 18. The isolated proteinmolecule of claim 12, wherein said amino acid sequence is (c).
 19. Anisolated protein molecule produced by the method comprising: (a)expressing the protein molecule of claim 18 from a host cell; and (b)recovering said protein molecule.
 20. The isolated protein molecule ofclaim 12, wherein said amino acid sequence is (d).
 21. An isolatedprotein molecule produced by the method comprising: (a) expressing theprotein molecule of claim 20 from a host cell; and (b) recovering saidprotein molecule.
 22. The isolated protein molecule of claim 12 furthercomprising a heterologous polypeptide.
 23. A composition comprising theprotein molecule of claim 12 and a pharmaceutically acceptable carrier.24. An isolated protein molecule comprising an amino acid sequenceselected from the group consisting of: (a) amino acids residues n to+193 of SEQ ID NO:2, wherein n is an integer in the range of −23 to +10;(b) amino acids residues −22 to m of SEQ ID NO:2, wherein m is aninteger in the range of +154 to +192; (c) amino acids n to m of SEQ IDNO:2, wherein n is an integer in the range of −23 to +10 and m is aninteger in the range of +154 to +192; and (d) a polypeptide consistingof a portion of the complete amino acid sequence encoded by the cDNAclone contained in ATCC Deposit No. 97148 wherein said portion excludesup to 33 amino acids from the amino terminus and up to 39 amino acidsfrom the C-terminus of said complete amino acid sequence.
 25. Theisolated protein molecule of claim 24, wherein said amino acid sequenceis (a).
 26. The isolated protein molecule of claim 24, wherein saidamino acid sequence is (b).
 27. The isolated protein molecule of claim24, wherein said amino acid sequence is (c).
 28. The isolated proteinmolecule of claim 24, wherein said amino acid sequence is (d).
 29. Anisolated protein molecule produced by the method comprising: (a)expressing the protein molecule of claim 24 from a host cell; and (b)recovering said protein molecule.
 30. The isolated protein molecule ofclaim 24 further comprising a heterologous polypeptide.
 31. Acomposition comprising the protein molecule of claim 24 and apharmaceutically acceptable carrier.
 32. An isolated protein moleculecomprising a first amino acid sequence having a least 95% identity to asecond amino acid sequence selected from the group consisting of: (a)amino acids +1 to +193 of SEQ ID NO:2; (b) a mature portion of theprotein encoded by the cDNA contained in ATCC Deposit No. 97148; (c)amino acids −23 to +193 of SEQ ID NO:2; and (d) the full length aminoacid sequence encoded by the cDNA contained in ATCC Deposit No. 97148,wherein % identity is determined using the Bestfit program withparameters that calculate % identity over the full length of said secondamino acid sequence and that allow for gaps of homology of up to 5% ofthe total number of amino acid residues in said second amino acidsequence.
 33. The isolated protein molecule of claim 32, wherein saidsecond amino acid sequence is (a).
 34. An isolated protein moleculeproduced by the method comprising: (a) expressing the protein moleculeof claim 33 from a host cell; and (b) recovering said protein molecule.35. The isolated protein molecule of claim 32, wherein said second aminoacid sequence is (b).
 36. An isolated protein molecule produced by themethod comprising: (a) expressing the protein molecule of claim 35 froma host cell; and (b) recovering said protein molecule.
 37. The isolatedprotein molecule of claim 32, wherein said second amino acid sequence is(c).
 38. An isolated protein molecule produced by the method comprising:(a) expressing the protein molecule of claim 37 from a host cell; and(b) recovering said protein molecule.
 39. The isolated protein moleculeof claim 32, wherein said second amino acid sequence is (d).
 40. Anisolated protein molecule produced by the method comprising: (a)expressing the protein molecule of claim 39 from a host cell; and (b)recovering said protein molecule.
 41. The isolated protein molecule ofclaim 32 further comprising a heterologous polypeptide.
 42. Acomposition comprising the protein molecule of claim 32 and apharmaceutically acceptable carrier.
 43. An isolated protein moleculecomprising a first amino acid sequence having at least 95% identity to asecond amino acid sequence selected from the group consisting of: (a)amino acids residues n to +193 of SEQ ID NO:2, wherein n is an integerin the range of −23 to +10; (b) amino acids residues −22 to m of SEQ IDNO:2, wherein m is an integer in the range of +154 to +192; (c) aminoacids n to m of SEQ ID NO:2, wherein n is an integer in the range of −23to +10 and m is an integer in the range of +154 to +192; and (d) apolypeptide consisting of a portion of the complete amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No. 97148 whereinsaid portion excludes up to 33 amino acids from the amino terminus andup to 39 amino acids from the C-terminus of said complete amino acidsequence, wherein % identity is determined using the Bestfit programwith parameters that calculate % identity over the full length of saidsecond amino acid sequence and that allow for gaps in homology of up to5% of the total number of amino acids residues in said second amino acidsequence.
 44. The isolated protein molecule of claim 43, wherein saidamino acid sequence is (a).
 45. The isolated protein molecule of claim43, wherein said amino acid sequence is (b).
 46. The isolated proteinmolecule of claim 43, wherein said amino acid sequence is (c).
 47. Theisolated protein molecule of claim 43, wherein said amino acid sequenceis (d).
 48. An isolated protein molecule produced by the methodcomprising: (a) expressing the protein molecule of claim 43 from a hostcell; and (b) recovering said protein molecule.
 49. The isolated proteinmolecule of claim 43 further comprising a heterologous polypeptide. 50.A composition comprising the protein molecule of claim 43 and apharmaceutically acceptable carrier.